US20100248257A1 - Compiled Methods for Analysing and Sorting Samples - Google Patents

Compiled Methods for Analysing and Sorting Samples Download PDF

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US20100248257A1
US20100248257A1 US12/647,747 US64774709A US2010248257A1 US 20100248257 A1 US20100248257 A1 US 20100248257A1 US 64774709 A US64774709 A US 64774709A US 2010248257 A1 US2010248257 A1 US 2010248257A1
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mhc
sample
cells
specific
labeled
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Kivin Jacobsen
Henrik Pedersen
Liselotte Brix
Jesper Lohse
Jørgen Schøller
Tina Jakobsen
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Dako Denmark ApS
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70503—Immunoglobulin superfamily
    • C07K14/70539—MHC-molecules, e.g. HLA-molecules
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/56—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule
    • A61K47/61—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule the organic macromolecular compound being a polysaccharide or a derivative thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
    • A61K47/64—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent
    • A61K47/6425—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent the peptide or protein in the drug conjugate being a receptor, e.g. CD4, a cell surface antigen, i.e. not a peptide ligand targeting the antigen, or a cell surface determinant, i.e. a part of the surface of a cell
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
    • A61K47/66—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid the modifying agent being a pre-targeting system involving a peptide or protein for targeting specific cells
    • A61K47/665—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid the modifying agent being a pre-targeting system involving a peptide or protein for targeting specific cells the pre-targeting system, clearing therapy or rescue therapy involving biotin-(strept) avidin systems
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B82—NANOTECHNOLOGY
    • B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
    • B82Y5/00—Nanobiotechnology or nanomedicine, e.g. protein engineering or drug delivery
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the present invention relates to methods for assaying an entity present in a sample.
  • the method comprises a number of individual steps, such as the steps of obtaining a sample, preparing the sample for assaying, and assaying the sample.
  • the method can comprise further optional processing steps associated with either sample processing or processing of data obtained as a result of having assayed the sample.
  • the sample can initially be assayed e.g. for presence of one or more entities.
  • the step of assaying the sample can further comprise the step of analyzing one or more characteristics of the one or more entities.
  • the method can comprise one or more further processing steps, such as data processing steps and/or sample processing steps associated with partitioning and/or isolation of the one or more entities in the sample which was subjected to the assaying procedure.
  • Analytical principles include binding to a solid support e.g affinity columns, flow cytometry and stationary cytometry such as immunohistochemistry.
  • a solid support e.g affinity columns, flow cytometry and stationary cytometry such as immunohistochemistry.
  • flow cytometry analyses often include reagents that have not been properly design to achieve the best possible result:
  • the individual reagents and their binding affinity or target abundance is not seriously considered and corrected for, and likewise, the fluorochromes chosen are not well enough separated as regards wavelength of maximum emission, and therefore, the spectra of the fluorochormes significantly overlap, blurring the data further.
  • the present invention relates to the fields of analysis, diagnostics, prognostics, standardization, characterization and enumeration of entities such as biological cells, as well as therapeutical applications.
  • the present invention in preferred aspects is directed to methods for the detection and/or analysis and/or isolation and optionally further manipulation of entities, such as cells, particles, and supra-molecular structures.
  • a method for detecting and/or analyzing and/or partitioning one or more entities potentially present in a sample comprising the steps of
  • the method can comprise the further steps of D) data processing and/or sample processing, such as sample analysis and/or partitioning, as well as E) data interpretation and/or further sample manipulations, such as cultivation and/or expansion of partitioned and/or isolated cells.
  • MHC molecules and MHC-multimers are complexes comprising multiple MHC molecules, such as MHC-peptide complexes comprising said MHC molecules.
  • FIG. 1 Example cell-antigens, for which marker molecules is antibodies or other molecules able to specifically bind the cell-antigen.
  • the table primarily shows cell-antigens able to differentiate blood cell populations. The cell-antigens are grouped for the cell type by which they are expressed. Also is shown which preferable antigens markers can be directed against and then used as contra or “dump” markers or as pro selective markers in gating strategies when analyzing samples with MHC multimers. The two columns at the right show cell-antigens important for identification of cell-“status”, e.g. activation, differentiation state.
  • FIG. 2 Gating strategy for identification and enumeration of antigen specific cytotoxic T cells
  • the method used in this example is a “No lyse” method, meaning that the sample red blood cells (RBC) is not lysed during cell preparation resulting in poorly defined scatter picture. Therefore the “RAW cell gate” is poorly defined in scatter parameters.
  • the scatter gated cells are further “purified” by a anti-CD3, anti-CD8 gate, transferring only the CD3 and CD8 positive cells, to the histogram/dot plot where the MHC CMV positive cells can be read off.
  • the labeling molecules used for the markers, anti-CD45, anti-CD3, anti-CD8 antibodies and for the MHC dextramers are, Cascade Yellow (CY), Pasific Blue (PB), Alexa700 and PE, respectively.
  • Counting beads was added to the sample and can be selected in different ways when analyzing the samples, e.g. using that the bead has low FSC compared to cells, or that the beads have high fluorescence signals detectable with Cascade yellow and FITC detectors. In this example the beads was selected for analysis by using the FITC detector.
  • Gate R8 gives the bead count in the acquired part of the sample and from this number the volume of sample acquired may be calculated.
  • the gates are defined as follows: R8 define the beads, and is subtracted out in all gates used except in Gate1.
  • Gate 1(R1) define the Raw cell gate in FSC/SSC plot, Gate 2, define the cells that also have High Alexa 700 and PB signals, i.e are CD8 and CD3 positive, Gate 3; restricts the CD8 positive cells between which the CMV specific cytotoxic T cells will be found.
  • FIG. 3 Scatter defined blood cell populations
  • Using a Lyse and wash procedure on peripheral blood samples enables the use of the two Scatter parameters, FSC and SSC to define the 3 major cell populations in blood.
  • Medium FSC, low SSC define the lymphocyte population
  • High FSC medium SSC defines the monocyte population
  • Medium FSC and high SSC defines the Granulocyte population of blood cells.
  • FIG. 4 Using one parameter (label) to contra-select for two different populations of entities
  • CD14 and CD15 antibodies (Qdot800), to contra-select the monocytes and the granulocytes from the analysis of the lymphocyte population.
  • FIG. 5 Spectral overlap between the label FITC into the detector dedicated to measure RPE
  • the emission spectra of a fluorochrome label may overlap with the spectra of another fluorochrome label (e.g. RPE).
  • a fluorochrome label e.g. FITC
  • RPE fluorochrome label
  • some of the signal from one label is detected and added to the signal of the other label measured in the detector for the second fluorochrome (CD3/RPE).
  • CD3/RPE fluorochrome label
  • subtraction of the signal from the other label has to be performed. It is therefore needed to know how much the signal from one label (FITC) is detected in the detector of the other label (RPE dedicated detector) (as illustrated).
  • the slope of the straight line is the spillover coefficient for FITC into the RPE dedicated detector: (101-39.8)/(225-91.4) ⁇ 45% of the FITC signal measured in the FITC dedicated detector is added to the signal measured in the RPE dedicated detector.
  • This coefficient can be determined for all fluorochromes using single stained samples, or mixed samples where each entity only is labeled with one fluorochrome. Using linear algebra on the intensity measured on each entity the spillover can be subtracted from each signal, a process called compensation.
  • FIG. 6 A basic lymphocyte phenotyping
  • Peripheral blood was assayed. The data shown are selected for the lymphocyte population using scatter parameters, and contra-selection for monocytes and granulocytes as illustrated in FIG. 4 .
  • the following detections molecules are, as shown: anti-CD3/FITC antibody, anti-CD56/RPE antibody, anti-CD4/Qdot705TM antibody, anti-CD8/Qdot800TM antibody, anti-CD20/APC antibody, anti-CD19/Qdot605TM antibody, and anti-CD5/Qdot655TM antibody.
  • Spillover has been determined between all labels, as illustrated in FIG. 5 , and the data has been compensated for that spillover for each cell, and plotted.
  • the intensity scale of each label (detection molecule) is shown on the axes.
  • FIG. 7 Example of exclusion gating
  • the data shown illustrate the use of exclusion gating. Entities that are defined as having very low FSC and very low SSC, are marked on the scatter plot, and cells being all cells not defined by this region is the cells of interest. This is an example of how to use parameters to reduce the amount of data, entity measurements that are saved in the file.
  • FIG. 8 Counter selection of cells using a DNA stain
  • the sample was stained with 7-AAD, a DNA stain that can not enter into viable cells but can enter necrotic and dead cells. Thus viable cells will be negative, while necrotic and dead cells will be positive for this detection molecule.
  • the viable cell data can now be further processed and/or the amount of dead cells present in the sample calculated. This is also an example of a label and marker being the same molecule.
  • FIG. 9 Detection of antigen specific T cells using MHC multimer constructs simultaneously with activation and intracellular staining of cytokines.
  • the figures illustrate IFN- ⁇ versus Pentamer staining of live lymphocytes I a blood sample from a human donor.
  • PBMCs were incubated with either a negative control (non-specific) Pentamer (A*0201/EBV (GLCTLVAML)) or a Pentamer specific for the cells of interest (B*0801/EBV (RAKFKQLL)), then stimulated with LAC (non-specific activation) or B*0801/EBV peptide (specific peptide activation) for 15 hours in the presence of Brefeldin A. Fixation, permeabilization and staining for IFN- ⁇ were carried out exactly as detailed in the protocol in example 55.
  • FIG. 10 Detection of Borrelia specific T cells in sample from human donor.
  • Human peripheral blood lymphocytes were ficoll purified and stained with either of a pool of MHC/APC dextramer molecule constructs containing peptides derived from Borrelia antigen Osp A and FlaB or the cells were stained with the negative control construct HLA-A*0201/GLAGDVSAV/APC. Both samples were also stained with the antibodies mouse-anti-human CD3/PB (clone UCHT1 from Dako), mouse-anti-human CD4/FITC (clone MT310 from Dako) and mouse-anti-human CD8/PE (clone DK25 from Dako). Following staining the sample was analyzed on a CyAn flow cytometer.
  • Live-gated, CD4 negative and CD3 positive lymphocytes are shown.
  • Dot plots showing live gated CD3 + /CD4 ⁇ lymphocytes from Borrelia patient stained with (A) Negative Control MHC Dextramer (HLA-A*0201(GLAGDVSAV) or (B) pool of MHC Dextramers containing peptides from Borrelia antigen Osp A and Fla B pool of MHC Dextramers containing peptides from Borrelia antigen are shown.
  • FIG. 11 HLA-typing using HLA-specific antibodies.
  • Human peripheral blood lymphocytes were ficoll purified from two donors, donor 1 and donor 2. Samples of cells from each donor were stained with PE-labeled anti-HLA-A*02 and anti-HLA-A*03 antibodies respectively and then analyzed on a flow cytometer. As a control unstained cell samples from each donor was also analyzed. Cells were gated using a lymphocyte gate in a FCS/SSC plot and the presence of PE positive staining was determined in each sample. As shown by the histogram plots in the figure donor 1 was positive for HLA-A*02 and negative for HLA-A*03 and donor 2 was negative for HLA-A*02 and positive for HLA-A*03.
  • FIG. 12 Gating strategy for no-lyse no-wash procedure.
  • CD45/PB antibody was used to set a trigger discriminator to allow the flow cytometer to distinguish between red blood cells and stained white blood cells. This was done during data collection by gating on CD45/PB positive cells in a CD45/PB vs. side scatter dot plot as shown in A. After data collection and during data analysis CD3 positive cells were selected by gating CD3/FITC positive cells in a CD3/FITC vs side scatter plot as shown in B. The final data was illustrated in a MHC multimer/PE vs CD8/APC plot (see FIG. 13 ).
  • FIG. 13 Identification of CMV-specific T cells in a blood sample using no-lyse no-wash procedure
  • Donor 1 was stained with a MHC multimer consisting of PE-conjugated 270 kDa dextran coupled with HLA-A*0201 in complex with beta2microglobulin and the peptide NLVPMVATV derived from Human Cytomegalo Virus (HCMV) (left panel) and with a negative control MHC multimer consisting of PE conjugated 270 kDa dextran coupled with HLA-A*0201 in complex with beta2microglobulin and the peptide ILKEPVHGV derived from Human Immunodeficiency Virus (HIV) (right panel).
  • MHC multimer consisting of PE-conjugated 270 kDa dextran coupled with HLA-A*0201 in complex with beta2microglobulin and the peptide NLVPMVATV derived from Human Cytomegalo Virus (HCMV) (left panel)
  • HCMV Human Cytomegalo Virus
  • Donor 2 was stained with a MHC multimer consisting of PE-conjugated 270 kDa dextran coupled with HLA-A*0101 in complex with beta2microglobulin and the peptide VTEHDTLLY derived from Human Cytomegalo Virus (HCMV) (left panel) and a negative control MHC multimer consisting of PE-conjugated 270 kDa dextran coupled with HLA-A*0101 in complex with beta2microglobulin and the peptide IVDCLTEMY derived from ubiquitin specific peptidase 9 (USP9) (right panel).
  • MHC multimer consisting of PE-conjugated 270 kDa dextran coupled with HLA-A*0101 in complex with beta2microglobulin and the peptide IVDCLTEMY derived from ubiquitin specific peptidase 9 (USP9) (right panel).
  • Donor 3 was stained with two MHC multimers consisting of PE conjugated 270 kDa dextran coupled with HLA-B*0207 in complex with beta2microglobulin and either of the peptides TPRVTGGGAM (left panel) or RPHERNGFTVL (center panel) both derived from Human Cytomegalo Virus (HCMV) and with a negative control MHC multimer consisting of PE-conjugated 270 kDa dextran coupled with HLA-B*0207 in complex with beta2microglobulin and the peptide TPGPGVRYPL derived from Human Immunodeficiency Virus (HIV) (right panel). All samples were also added Anti-CD45/PB, anti-CD3/FITC and anti-CD8/APC antibodies. The samples were gated as shown in FIG. 12 .
  • FIG. 14 Enumeration of specific T cells using CytoCountTM beads.
  • Anti-CD45/CY anti-CD3/APC and anti-CD8/PB antibody was added together with 50 ⁇ l CytoCount beads (1028 beads/ ⁇ l). Following staining for 15 minutes PBS was added to 1 ml and the samples analyzed on a CyAn flow cytometer. During analysis CD45/CY antibody was used to set a trigger discriminator to allow the flow cytometer to distinguish between red blood cells and stained white blood cells and CD3/APC antibody was used to gate for CD3 positive T lymphocytes.
  • Amount of counted beads in sample A are shown in the histogram C and amount of beads counted in the negative control sample B are show in histogram D.
  • HLA-A*0101(VTEHDTLLY) specific T cells in the blood sample was determined as follows:
  • FIG. 15 Flow chart showing the process of measurement of antigen reactive T-Cells by IFN- ⁇ capture in blood samples by ELISPOT assay.
  • an antibody means an isolated or recombinant binding agent that comprises the necessary variable region sequences to specifically bind an antigenic epitope. Therefore, an antibody is any form of antibody or fragment thereof that exhibits the desired biological activity, e.g., binding the specific target antigen.
  • Antibodies can derive from multiple species. For example, antibodies include rodent (such as mouse and rat), rabbit, sheep, camel, and human antibodies. Antibodies can also include chimeric antibodies, which join variable regions from one species to constant regions from another species.
  • antibodies can be humanized, that is constructed by recombinant DNA technology to produce immunoglobulins which have human framework regions from one species combined with complementarity determining regions (CDR's) from a another species' immunoglobulin.
  • the antibody can be monoclonal or polyclonal.
  • Antibodies can be divided into isotypes (IgA, IgG, IgM, IgD, IgE, IgG1, IgG2, IgG3, IgG4, IgA1, IgA2, IgM1, IgM2)
  • antibody refers to an intact antibody, or a fragment of an antibody that competes with the intact antibody for antigen binding.
  • antibody fragments are produced by recombinant DNA techniques.
  • antibody fragments are produced by enzymatic or chemical cleavage of intact antibodies.
  • Exemplary antibody fragments include, but are not limited to, Fab, Fab′, F(ab′)2, Fv, and scFv.
  • Exemplary antibody fragments also include, but are not limited to, domain antibodies, nanobodies, minibodies ((scFv-CH 3 ) 2 ), maxibodies ((scFv-CH 2 —CH 3 ) 2 ), diabodies (noncovalent dimer of scFv).
  • Antigen Substance that cause an immune response
  • Antigen presenting cell An antigen-presenting cell (APC) as used herein is a cell that displays foreign antigen complexed with MHC on its surface.
  • Antigenic peptide Any peptide molecule that is bound or able to bind into the binding groove of either MHC class 1 or MHC class 2.
  • CMV Cytomegalo Virus
  • Counting beads Beads or particles that can be used as internal control beads enabling absolute cell count in a sample.
  • Detection molecule A molecule or a complex comprising a marker molecule and a labeling molecule.
  • a detection molecule can be both in one molecule.
  • Hapten a residue on a molecule for which there is a specific molecule that can bind, such as an antibody.
  • Fluorescein in addition of being a fluorochrome, it may also work as a hapten, for which there is an antibody that can bind specifically.
  • Labeling molecule Used interchangeably with label herein.
  • Example labeling molecules are chromophores (e.g. coomassie blue), fluorochromes (e.g. FITC), radioactive molecules (e.g. molecules containing the radionuclide P-32 or S-35). Precipitated molecules exposing a hapten.
  • Marker molecule Used interchangeably with marker herein. A molecule that specifically associates covalently or non-covalently with a molecule belonging to or associated with an entity.
  • MHC Denotes the major histocompatibility complex.
  • a “MHC Class I molecule” as used everywhere herein is defined as a molecule which comprises 1-3 subunits, including a heavy chain, a heavy chain combined with a light chain (beta 2 m), a heavy chain combined with a light chain (beta 2 m) through a flexible linker, a heavy chain combined with a peptide, a heavy chain combined with a peptide through a flexible linker, a heavy chain/beta 2 m dimer combined with a peptide, and a heavy chain/beta 2 m dimer with a peptide through a flexible linker to the heavy or light chain.
  • the MHC molecule chain can be changed by substitution of single or by cohorts of native amino acids or by inserts, or deletions to enhance or impair the functions attributed to said molecule.
  • substitution of XX with YY in position nn of human beta 2 m enhance the biochemical stability of MHC Class I molecule complexes and thus can lead to more efficient antigen presentation of subdominant peptide epitopes.
  • MHC complex is herein used interchangeably with MHC-peptide complex, unless it is specified that the MHC complex is empty, i.e. is not complexed with peptide.
  • a “MHC Class II molecule” as used everywhere herein is defined as a molecule which comprises 2-3 subunits including an alpha-chain and a beta-chain (alpha/beta-dimer), an alpha/beta dimer with a peptide, and an alpha/beta dimer combined with a peptide through a flexible linker to the alpha or beta chain, an alpha/beta dimer combined through an interaction by affinity tags e.g. jun-fos, an alpha/beta dimer combined through an interaction by affinity tags e.g. jun-fos and further combined with a peptide through a flexible linker to the alpha or beta chain.
  • affinity tags e.g. jun-fos
  • affinity tags e.g. jun-fos
  • the MHC molecule chains can be changed by substitution of single or by cohorts of native amino acids or by inserts, or deletions to enhance or impair the functions attributed to said molecule. Under circumstances where the alpha-chain and beta-chain have been fused, to form one subunit, the “MHC Class II molecule” can comprise only 1 subunit.
  • MHC complexes and “MHC constructs” are used interchangably herein.
  • MHC protein and “MHC molecule” are used interchangably herein. Accordingly, a functional MHC peptide complex comprises a MHC protein or MHC molecule associated with a peptide to be presented for cells or binding partners having an affinity for said peptide.
  • MHC complexes and “MHC multimers” as used herein are meant such complexes and multimers thereof, which are capable of performing at least one of the functions attributed to said complex or multimer.
  • the terms include both classical and non-classical MHC complexes.
  • the meaning of “classical” and “non-classical” in connection with MHC complexes is well known to the person skilled in the art.
  • Non-classical MHC complexes are subgroups of MHC-like complexes.
  • MHC complex includes MHC Class I molecules, MHC Class II molecules, as well as MHC-like molecules (both Class I and Class II), including the subgroup non-classical MHC Class I and Class II molecules.
  • the MHC molecule can suitably be a vertebrate MHC molecule such as a human, a mouse, a rat, a porcine, a bovine or an avian MHC molecule.
  • a vertebrate MHC molecule such as a human, a mouse, a rat, a porcine, a bovine or an avian MHC molecule.
  • Such MHC complexes from different species have different names. E.g. in humans, MHC complexes are denoted HLA. The person skilled in the art will readily know the name of the MHC complexes from various species.
  • MHC dextramers MHC-peptide complexes coupled to dextran through covalent or non-covalent interactions.
  • the dextramer can furthermore comprise flourochrome or other label coupled to dextran molecules through covalent or non-covalent interactions.
  • MHC multimer The terms MHC multimer, MHCmer and MHC'mer herein are used interchangeably, to denote a complex comprising more than one MHC-peptide complexes, held together by covalent or non-covalent bonds.
  • MHC molecule herein denotes the empty MHC protein, i.e. MHC protein not complexed with antigenic peptide.
  • MHC multimer The terms MHC multimer, MHCmer and MHC'mer herein are used interchangeably, to denote a complex comprising more than one MHC-peptide complexes, held together by covalent or non-covalent bonds.
  • MHC tetramers are MHC multimers consisting of streptavidin/avidin associated with 4 biotinylated MHC molecules.
  • Negative control peptide peptide binding the MHC allele of choice, but that does not support binding of the resultant MHC-peptide complex to the desired TCR
  • RBC Red Blood cell
  • Vacutainer a registered brand of test tube specifically designed for venipuncture.
  • the tube has vacuum inside and a rubber cap in the top. The vacuum causes blood to move through the needle inserted into the rubber cap and into the tube.
  • entity refers to any cell, molecule, molecular complex, particle or sub-component, detectable or selectable by the use of marker molecules, and specific inherent characteristics.
  • Example entities are cells such as T-cells or B-cells, cell like particles, micelles and liposome's, beads, particles, polymer beads, supramolecular structures and supramolecular compartments.
  • the present invention in one embodiment is directed to the analysis and sorting (partitioning) of entities such as cells, supramolecular compartments and particles.
  • the analytical principles can include binding of the entities to e.g. affinity columns or other types of solid supports, flow cytometry or stationary cytometry, such as immunohistochemistry.
  • the entities to be assayed and/or sorted are MHC molecules.
  • the MHC molecules can be used in the assaying and/or sorting of the entities in question.
  • the MHC molecules can be MHC multimers as disclosed in more detail herein below.
  • the entities to be assayed are T cells or antigen presenting cells.
  • the T cells and/or antigen presenting cells can be used in the assaying.
  • Antigen presenting cells are herein any cell expressing MHC I molecules and/or MHC II molecules.
  • the present invention also includes analysis of substances released from entities e.g. soluble factors secreted from cells (cytokines, hormones ect.)
  • MHC molecules function as antigenic peptide receptors, collecting peptides inside the cell and transporting them to the cell surface, where the MHC-peptide complex may be recognized by T-lymphocytes.
  • MHC class I and II Two classes of classical MHC molecules exist, MHC class I and II. The most important difference between these two molecules lies in the protein source from which they obtain their associated peptides.
  • MHC class I molecules present peptides derived from endogenous antigens degraded in the cytosol and are thus able to display fragments of viral proteins and unique proteins derived from cancerous cells. Almost all nucleated cells express MHC class I on there surface even though the expression level varies among different cell types.
  • MHC class II molecules bind peptides derived from exogenous antigens.
  • MHC class II molecules are only expressed on professional antigen presenting cells like B cells and macrophages.
  • MHC class I molecules consist of two polypeptide chains, a heavy chain, a, spanning the membrane and a light chain, ⁇ 2-microglobulin ( ⁇ 2m).
  • the heavy chain is encoded in the gene complex termed the major histocompatibility complex (MHC), and comprises three domains, ⁇ 1, ⁇ 2 and ⁇ 3.
  • MHC major histocompatibility complex
  • the ⁇ 2m chain is not encoded in the MHC gene and consists of a single domain, which together with the ⁇ 3 domain of the heavy chain make up a folded structure that closely resembles that of the immunoglobulins.
  • the ⁇ 1 and ⁇ 2 domains pair to form the peptide binding cleft, consisting of two segmented a helices lying on a sheet of eight 3-strands.
  • MHC class I molecule In humans as well as in mice three different types of MHC class I molecule exist. HLA-A, B, C are found in humans while MHC class I molecules in mice are designated H-2K, H-2D and H-2L.
  • the MHC class II molecule is composed of two membrane spanning polypeptide chains, ⁇ and ⁇ , of similar size (about 30000 Da). Genes located in the major histocompatibility complex encode both chains. Each chain consists of two domains, where ⁇ 1 and ⁇ 1 forms a 9-pocket peptide-binding cleft, where pocket 1, 4, 6 and 9 are considered as major peptide binding pockets.
  • the ⁇ 2 and ⁇ 2, like the ⁇ 2 and ⁇ 2m in the MHC class I molecules, have amino acid sequence and structural similarities to immunoglobulin constant domains. In contrast to MHC class I molecules, where the ends of the peptide is buried, peptide-ends in MHC class II molecules are not.
  • HLA-DR, DQ and DP are the human class II molecules
  • H-2A, M and E are those of the mice.
  • MHC genes A remarkable feature of MHC genes is their polymorphism accomplished by multiple alleles at each gene.
  • the polygenic and polymorphic nature of MHC genes is reflected in the peptide-binding cleft so that different MHC molecules bind different sets of peptides.
  • the variable amino acids in the peptide binding cleft form pockets where the amino acid side chains of the bound peptide can be buried. This permits a specific variant of MHC to bind some peptides better than others.
  • MHC multimers Due to the short half-life of the peptide-MHC-T cell receptor ternary complex (typically between 10 and 25 seconds) it is difficult to label specific T cells with labelled MHC-peptide complexes.
  • MHC multimers have been developed. These are complexes that include multiple copies of MHC-peptide complexes, providing these complexes with an increased affinity and half-life of interaction, compared to that of the monomer MHC-peptide complex. The multiple copies of MHC-peptide complexes are attached, covalently or non-covalently, to a multimerization domain.
  • MHC multimers include the following:
  • MHC multimers will be used interchangeably with the terms MHC'mers and MHCmers, and will include any number, (larger than one) of MHC-peptide complexes, held together in a large complex by covalent or non-covalent interactions between a multimerization domain and one or more MHC-peptide complexes, and will also include the monomeric form of the MHC-peptide complex, i.e. a MHC-peptide complex that is not attached to a multimerization domain.
  • MHC multimers thus include MHC-dimers, MHC-tetramers, MHC-pentamers, MHC-hexamers, as well as organic molecules, cells, membranes, polymers and particles that display two or more MHC-peptide complexes.
  • Example organic molecule-based multimers include functionalized cyclic structures such as benzene rings; example cell-based MHC multimers include dendritic cells and antigen presenting cells (APCs); example membrane-based MHC multimers include liposomes and micelles carrying MHC-peptide complexes in their membranes; and example polymer-based MHC multimers include MHC-dextramers (dextran to which a number of MHC-peptide complexes are covalently or non-covalently attached).
  • any kind of multimerization domain may be used, including any kind of cell, polymer, protein or other molecular structure, or particles and solid supports.
  • the MHC multimers may be generated by first expressing and purifying the peptide components of the MHC protein, and then combining the MHC peptide components and the peptide, to form the MHC-peptide complex. Then an appropriate number of MHC-peptide complexes are linked together by covalent or non-covalent bonds to a multimerization domain.
  • reactive groups of the multimerization domain e.g. vinyl sulfone functionalities on a dextran polymer
  • reactive groups on the MHC protein e.g. amino groups on the protein surface
  • non-covalent interaction between a part of the MHC protein e.g. a biotinylated peptide component
  • the multimerization domain e.g. four binding sites for biotin on the strepavidin tetrameric protein.
  • the MHC multimer may be formed by the non-covalent association of amino acid helices fused to one component of the MHC protein, to form a pentameric MHC multimer, held together by five helices in a coiled-coil structure making up the multimerization domain.
  • the MHC multimers may be labeled with fluorochromes and used in flow cytometry.
  • MHC multimers may be used to label T-cells carrying specific T-cell receptors (TCR).
  • TCR T-cell receptors
  • the MHC multimers may be labeled with fluorochromes or enzymes, in order to specifically stain specific T-cells carrying TCR's that specifically bind the MHC multimer in question.
  • ELISA and ELISA-like analyses may be performed with MHC multimers that are labeled with enzymes, chromophors or fluorochromes.
  • MHC allele For all of the above applications, it is important to choose the right MHC allele as well as a peptide that binds well to the MHC protein. It is also important that the chosen MHC allele and peptide forms a MHC-peptide complex that is efficiently and specifically recognized by the TCR. For applications that involve binding as well as activation of cells, further restrictions on the choice of MHC and peptide may apply.
  • Marker molecules are molecules that specifically interact with target molecules on the surface or in the interior of entities such as cells or beads. Marker molecules can be any kind of molecule, for example antibodies, nucleic acids aptamers, small organic molecules and many other types of molecules. Marker molecules that are used in the analysis of cell populations include molecules that specifically bind to extra-cellular membrane proteins or associated molecules (e.g. receptors and glycosides), intracellular proteins and RNA, and DNA in the nucleus of eukaryotic cells. Particularly interesting marker molecules include so-called MHC-multimers. MHC-multimers are complexes comprising multiple MHC-peptide complexes.
  • Flowcytometry is a powerful method for which to analyze many entities for multiple parameters at the same time.
  • the principal of detecting a specific molecule in or on the surface of the entity is to have a specific marker molecule, such as an antibody recognizing the molecule in question.
  • the marker molecule is labeled with a fluorochrome, thus if the entity displays the specific target it is detected after laser light excitation by the flow cytometer as being fluorescing, and the amount of fluorescent light is proportional to the amount of marker molecules on the entity.
  • Many such pairs of markers and target molecules linked fluorochromes can be detected at the same time by flow cytometry.
  • the data are analyzed and interpret by selectively selecting the entities displaying certain fluorescents markers and or selecting entities lacking selective markers, a process called positive and negative or dump gating, respectively.
  • the identification of antigen specific cytotoxic T-cells are identified by the binging of specific MHC labeled molecules and that of labeled anti-CD8 antibody, as well as negative selected for the presence of CD4 identified by a labeled anti-CD4 antibody.
  • the same process can be performed on a cell sorter, where the cells that fulfill the gating criteria is physically sorted into a purified population of cells with the phenotype described by the gating strategy. Extending the analysis to encompass more marker molecules for a more precise phenotyping of the entities of interests, will need more labeling molecule, or maybe even automated help in the gating strategy as the gating becomes increasingly more requiring with more markers involved.
  • the full workflow from sample source and sampling to interpretation of the data is involving many different steps, which open possibility for mistakes, thus an easy and comprehensive methodology throughout the workflow is essential for reproducibility of data and reliable diagnosis of diseases.
  • the MHC multimer may be labeled with a tag that can be recognized by e.g. a secondary antibody, optionally labeled with HRP or another label.
  • the bound marker molecule is then detected by its fluorescence (for fluorochrome), or by addition of an enzyme-labeled antibody directed against the marker molecule, or another component of the molecule (e.g. one of the protein chains, a label on the multimerization domain, a chemical “tag or hapten”).
  • the enzyme may be Horse Raddish Peroxidase (HRP) or Alkaline Phosphatase (AP), both of which may catalyze a chemical conversion of a non-detectable to a precipitated detectable molecule such as a precipitation of a chromophore detectable by it absorption, or by the enzymatic generation of chemiluminescence. Or it may catalyze a precipitation of molecules exposing a tag or hapten for binding of additional detection molecule in situ.
  • HRP Horse Raddish Peroxidase
  • AP Alkaline Phosphatase
  • the colored deposit, or a label molecule bound to the exposed tag in the precipitate identifies the binding site of the marker molecule, and can be visualized by photometry, such as general microscopy, fluorescence microscopy or digital scanning and photographic techniques.
  • the marker molecule e.g. a MHC multimer can also be directly labeled with e.g. HRP or AP, or a fluorochrome, and used in IHC without an additional secondary binding molecule.
  • Antibodies are standard reagents used for staining of formalin-fixed tissue sections; some antibodies recognize linear epitopes and binds easily to fixed tissue, however other antibodies recognize conformal epitopes, or hidden epitopes in the fixed tissue, therefore, often an antigen retrieval procedure is applied, e.g heating the sample in a microwave oven, or other ways to shift the fixed conformation of the antigen.
  • MHC multimers are expected to recognize a conformational epitope on the TCR, thus a gently fixation or a antigen retrieval procedure might be required in most cases.
  • staining is performed on frozen tissue sections, and the fixation is done after MHC multimer staining.
  • Steps A, B and C are essential steps in so far as all samples are prepared for assaying and subsequently assayed, whereas steps D, E and F are optional and depends on the purpose and nature of the assaying method (or step) performed in (step) C.
  • steps are also crucial for the outcome of the experiments, and different starting points (i.e. sample source and sampling process, cf. step A above)) often necessitate different sample preparation steps (c.f. step B above) and the different analytical procedures subsequently applied (cf. step C, and optionally further steps D, E and F).
  • the analytical procedures preferably comprise method steps for analyzing and isolating entities by means of marker molecules, such as MHC molecules and MHC multimers, that are capable of binding specific target ligands present in or exposed on the entities of interest.
  • marker molecules such as MHC molecules and MHC multimers
  • Target ligands can be labelled.
  • the types of analyses which can be carried out in accordance with the present invention include cytometry (flow cytometry and stationary cytometry) and batch treatment.
  • the analytical procedures i.e. assaying step C) described herein below in more detail allow the analysis (quantization) or selection of entities, based on the characteristics of the individual entity.
  • characteristics of the entities can include e.g. the type of entity (e.g. is the entity a T-cell or a B-cell), as well as status of the entity (e.g. is the entity a yeast cell in phase G1, G2 or S; is the cell dead or viable).
  • step A to F each of the sub-processes (steps A to F) is described. Preferred sub-processes are indicated and several preferred combinations of sub-processes are described in detail.
  • Sampling is the process by which the sample is collected from the source and optionally kept until the sample can be prepared and analyzed.
  • Sample sources include samples obtained from live as well as non-live sources, including but not limited to humans, animals, birds, insects, plants, algae, fungi's, yeast, viruses, bacteria and phages, multi-cellular and mono-cellular organisms, and chemical reactions comprising e.g. supra-molecular structures, transitions state molecules, chemical, and enzymatic reactions.
  • Human and animal samples include blood, semen, and tissue samples.
  • Sources of pathogens, such as bacteria, viruses and other infective organisms and sources may be environmental samples such as drinking water, sewage, or soil.
  • Blood is a preferred sample source for flow cytometry.
  • Other preferred human sources for flow cytometry include bodily fluids such as urine, saliva, lymphatic fluid, cerebrospinal liquid and semen, as well as solid tissue samples.
  • tissue may be subjected to enzymatic digestion, or the tissue may be mechanically grinded or micro-dissected to yield suspensions suitable for flow cytometry.
  • solid samples may be fixed and paraffin embedded, or frozen and analyzed as thin sections on slides.
  • Liquid samples may be subjected to cytospin or immobilized by some other method on slides, prior to stationary cytometry analysis.
  • Animal samples may be subjected to similar procedures.
  • the sample may be collected into a container that is appropriate for the intended type of analysis, or that protects the sample from the environment or protects the experimenter from the sample.
  • the sampling devise preferably is a sterile, single-use devise, into which the sample passively drips or is actively drawn by e.g. vacuum.
  • Such devises may hold up to one liter or more of blood; for analytical purposes as little as a few milliliters or less may be collected. Even smaller volumes may be obtained through micro-puncture followed by aspiration of e.g. a single drop of blood directly into the analytical devise.
  • the sampling devise is preferably designed so as to allow sampling by non-professional health workers or the patient.
  • the sample is drawn directly from patient circulation, e.g. during surgery, which may allow simultaneous analysis in a continuous manner.
  • sample containers include mechanical strength to allow centrifugation, transparent walls or opaque walls protecting the sample from light with transparent windows to allow visual sample monitoring.
  • the sampling needle should be easily extractable preferably directly into disposal containers.
  • a chemical or electronic monitoring devise that integrates time and temperature following sample introduction into the devise may be used as a measure of sample integrity.
  • the sample is collected into a container under circumstances where the volume can be easily determined or standardized; for this purpose a vacutainer is particularly preferable, as they are easy of use, and draw a specific quantity of blood.
  • a vacutainer is particularly preferable, as they are easy of use, and draw a specific quantity of blood.
  • Other preferred containers include, open blood glass, donor blood bag, and capillary tubes.
  • Example containers include microtiter plates, sample tubes for large, e.g. 1-20 ml, or small, e.g. 10 ⁇ l-1 ml sample volume, and may be handled individually or in a dedicated rack system.
  • the sampling devise may contain anti-oxidants, anti-coagulants, biocides or other reagents prior to the addition of the sample. These reagents serve to protect the sample from the surroundings, or alternatively, protect the surroundings (e.g. the experimenter) from the sample.
  • Example reagents are anti-coagulants such as citrate, EDTA or heparin; fixatives such as formaldehyde or glutardialdehyde; various preservatives such as azide, benzoate and or biocides against e.g. HIV virus or other viruses, or against bacteria.
  • the reagents thus serve to maintain sample integrity until it can be prepared for, analyzed.
  • the sample device may contain reagents that are employed during the analytical step (e.g. marker molecules such as MHC-dextramers, antibodies, fluorescent beads or other probes and reference material to be used for the subsequent sample analyses).
  • the reagents may be added during sample preparation (see below).
  • the sampling devise may also comprise mechanical devises such as filters, additional inlets and outlets that allow reagents to be added and samples extracted; solutions that by centrifugation establish a density gradient, and other features that are part of the sample preparation process.
  • the sample may be homogenized or filtered, or may be solubilized by addition of e.g. proteases and nucleases, in order to perform e.g. a flow cytometry analysis.
  • the primary sample volume and concentration of added reagents are accurately known.
  • Internal control reagents such as e.g. beads, optionally encoded and/or of known quantity and/or signal intensity (e.g. fluorescence intensity), may be added to the primary sample or to the sampling devise prior to sampling.
  • These internal control reagents may serve as a means for quantitation of specific entities, analyzed volume of sample, effect of sample preparation and storage, or fixation (if performed) on the sample, as a standard for the assay performed, e.g. fluorescent intensity standard in cytometry.
  • beads added to the sample could serve as a mean for identification of the sample at a later stage, as a measure of sample loss during the sample preparation or analysis steps that follow.
  • Beads may be encoded by their fluorescence, either by the bead's intensity and/or by its fluorescence or multiple fluorescence's , or by its physical properties such as forward or side scatter in fluorescence-based flow cytometry.
  • RFID Radio Frequency IDentification
  • graphical or optical labels may be used to encode the beads.
  • the bead encoding scheme should ideally establish an unequivocal information link between sample origin, e.g. patient identity, time of extraction and identity of the health professional who acquired the sample.
  • the sample is transferred to a sampling devise, containing reagents that ensure sample stability and allow efficient sample preparation, where said sampling devise fits directly into a fully automated instrument for data acquisition.
  • the sample may be applied to the analytical instrument manually, or by the use of a robotic or automated device, that handles tubes from a rack and loads the sample on the instrument.
  • Other handling devices may be used, such as a tube carrousel, tubes in an X/Y pattern rack or linear rack, and microtiter-, or multiwell plates, depending on the application and instrument used.
  • samples may be collected from diverse sources such as human blood or tissue, environmental samples or chemical reactions. Often, additional steps must be performed in order to run the analysis on the sample.
  • This process is called sample preparation, and include i) addition of anti-oxidants, anti-coagulants, biocides or other reagents that protect the sample from the surroundings, or alternatively, protect the surroundings (e.g. the experimenter) from the sample, ii) physical or chemical removal or destruction of troublesome contaminants, iii) addition of chemicals or processing steps that provide the sample in a form (e.g. solid or fluid) appropriate for the analytical step that follows, iv) enrichment of the desired entities, v) labeling of the entities, and vi) addition of internal standard reagents.
  • a form e.g. solid or fluid
  • Preparation of the sample for subsequent analysis can be an integrated part of the total analysis, because the way the sample preparation is performed will influence the basic of the subsequent analysis. Sample preparation may remove or add factors, and therefore influences the analytical data acquired.
  • the sample may be fractionated to enrich for or remove certain factors or entities. This may be done by physical or chemical separation.
  • Example physical separations are simple centrifugation, density centrifugation in ficoll, glycerol, sucrose or other chemicals that alter the physical density of the sample during centrifugation. Centrifugation is often used on blood to separate different cell types; typical cell types that may be separated include red blood cells (RBC), and lymphocyte from mono- and granulocytes.
  • RBC red blood cells
  • lymphocyte from mono- and granulocytes.
  • An example chemical treatment is induced precipitation or destruction of contaminants or undesired entities by an added chemical.
  • chemical separation include selective destruction of entities, e.g. lysis of RBC in full blood using chemical reagents, such as UtiLyseTM, EasyLyseTM, FACSLyseTM, or other ammonium containing solutions followed by osmotic shock.
  • chemical reagents such as UtiLyseTM, EasyLyseTM, FACSLyseTM, or other ammonium containing solutions followed by osmotic shock.
  • Contaminants may be removed by binding to immobilized marker molecules such as antibodies, or subpopulations of entities may be enriched by binding a sub-population of entities to solid support, for example carrying immobilized marker molecules.
  • This type of enrichment can be used to remove undesired entities, in which case the solid support with the immobilized entities is simply discarded, or it can be used to isolate a subpopulation containing the desired entities, in which case the non-bound entities are first removed, and then the immobilized entities released from solid support.
  • Internal standard reagents may be added during sample preparation. As an example, quantitation such as determination of concentration, labeling efficiency, signal intensity or other characteristics of specific entities is often more accurate when employing an added, internal standard reagent.
  • Example internal standard reagents include: i) a known number of particles (for quantization of amount of entities), ii) particles with known fluorescence intensity, and iii) particles capable of associating with a specific amount of a labeling dye.
  • a known number of internal control beads may be added to a known volume of the primary sample initially. This will allow for a correction for loss of material during sample preparation after the analysis, provided that the numbers of internal control beads are determined during the analytical step.
  • Such beads are in the following referred to as counting beads.
  • the counting beads are microparticles with scatter properties that put them in the context of the cells of interest when registered by a flow cytometer. They can be either labelled with antibodies, fluorochromes or other marker molecules or they may be unlabelled.
  • the beads are polystyrene beads with molecules embedded in the polymer that are fluorescent in most channels of the flow-cytometer.
  • counting bead and microparticle are used interchangeable.
  • the counting beads employed in the methods and compositions described here should preferably be small, and are preferably between 0.1 ⁇ m and 100 ⁇ m, preferably about 5 ⁇ m in diameter.
  • the microparticles should preferably be made of such material and be of such size as to stay suspended, with minimal agitation if necessary, in solution or suspension (i.e., once the sample is added). It should preferably not settle any faster than the cells of interest in the sample.
  • the material from which the microparticles are made should be such as to avoid clumping or aggregation, i.e., the formation of doublets, triplets, quadruplets and other multiplets.
  • a final count of counting beads of at least 1000/ ⁇ l is preferred but any number can be counted.
  • the counting beads should preferably be labelled with a reporter molecule, such as a fluorescent molecule (which is described in further detail elsewhere).
  • a microparticle which is autofluorescent may be employed.
  • Counting beads may be selected from the group consisting of fixed chicken red blood cells, coumarin beads, liposomes containing a fluorescent dye, fluorescein beads, rhodamine beads, fixed fluorescent cells, fluorescent cell nuclei, microorganisms and other beads tagged with a fluorescent dye.
  • compact particles such as agarose beads, polyacrylamide beads, polystyrene beads, silica gel beads, etc.
  • Beads or microparticles suitable for use include those which are used for gel chromatography, for example, gel filtration media such as Sephadex.
  • Suitable microbeads of this sort include Sephadex G-10 having a bead size of 40-120 ⁇ (Sigma Aldrich catalogue number 27, 103-9), Sephadex G-15 having a bead size of 40-120 ⁇ m (Sigma Aldrich catalogue number 27, 104-7), Sephadex G-25 having a bead size of 20-50 ⁇ m (Sigma Aldrich catalogue number 27, 106-3), Sephadex G-25 having a bead size of 20-80 ⁇ m (Sigma Aldrich catalogue number 27, 107-1), Sephadex G-25 having a bead size of 50-150 ⁇ m (Sigma Aldrich catalogue number 27,109-8), Sephadex G-25 having a bead size of 100-300 ⁇ m (Sigma Aldrich catalogue number 27, 110-1), Sephadex G-50 having a bead size of 20-50 ⁇ m (
  • Sepharose beads for example, as used in liquid chromatography, may also be used.
  • Q-Sepharose, S-Sepharose and SP-Sepharose beads available for example from Amersham Biosciences Europe GmbH (Freiburg, Germany) as Q Sepharose XL (catalogue number 17-5072-01), Q Sepharose XL (catalogue number 17-5072-04), Q Sepharose XL (catalogue number 17-5072-60), SP Sepharose XL (catalogue number 17-5073-01), SP Sepharose XL (catalogue number 17-5073-04) and SP Sepharose XL (catalogue number 117-5073-60) etc.
  • plastic microbeads are usually solid, they may also be hollow inside and could be vesicles and other microcarriers. They do not have to be perfect spheres in order to function in the methods described here.
  • Plastic materials such as polystyrene, polyacrylamide and other latex materials may be employed for fabricating the beads, but other plastic materials such as polyvinyl chloride, polypropylene and the like may also be used.
  • Polystyrene is a preferred material.
  • the microparticles include unlabelled beads, beads with antibodies, fluorochromes or other small molecules conjugated to the surface or beads with fluorochromes embedded in the polymer.
  • internal control reagents with known characteristics, e.g. known fluorescence intensity, allow correction of e.g. an instrument's drift as regards fluorescence measurement.
  • marker molecules may be added.
  • marker molecules may be added first, and allowed to bind the entities first, and then labeling molecules that associate with the marker molecules may be added. Entities that bind these marker molecules in this way become labeled (stained), which will allow their identification and/or isolation in the subsequent analysis step.
  • the marker molecules may be added manually or by an automated pipetting robot. Marker molecules and labeling are described in further detail elsewhere herein.
  • the other reagents may be added to the sample as an optimized mix of different marker molecules.
  • the optimized combination and amount of reagents are immobilized in a matrix of sugars in a container, such as a tube or well of a microtiter plate. Then the sample (e.g. “untreated full blood”, diluted blood, or “lyse no wash”, or “lyse washed” blood) is added into the container and mixed with the immobilized content of the container.
  • Cells in a collected sample may be stimulated to secrete soluble factors, express new and/or increased levels of surface receptors and/or stimulated to proliferate.
  • Cells can be stimulated by addition of antigen and/or antigenic peptide and/or addition of cytokines.
  • Cells may also be stimulated by addition of antigen presenting cells either alone or in combination with either of the above describes stimulators.
  • cells in a sample can be stimulated by addition of antibody, antibody fragments, protein ligands or other molecules whos interaction with specific receptors on the surface of the cell result in stimulation.
  • Other stimulators include but is not limited to Lipopolysaccharide, phytohaemagglutinin (PHA), Con A, Pokeweed mitogen, other mitogens, chemicals, small organic molecules or any other substances able to stimulate cells.
  • Sample preparation for stationary cytometry is here exemplified by a typical sample preparation process for paraffin embedded samples for use in immunohistochemistry
  • Tissue is remove from the donor or patient, and the tissue is immediately fixated in formalin. After fixation the tissue is embedded in paraffin, which allow for storage of the tissue for years.
  • Sample preparation starts by cutting thin sections of the paraffin block, an mount the sections onto glass plates.
  • the sample is deparafinated by incubation in organic solvents, and optionally stained with different chemical dyes, staining different compartments of the cells, e.g nucleus and the lipid membrane.
  • an antigen retrieval procedure is required for subsequent binding of certain antibodies.
  • the sample can now be stained with a detection molecule, e.g. an antibody or other marker molecules linked to a fluorochrome or an enzyme for its detection.
  • the sample is incubated with a marker molecule that is labeled in a subsequent process by an anti-antibody or another marker molecule linked to a label molecule, such as an HRP, or AP (Horse raddish peroxidase, or alkaline phosphatase).
  • a marker molecule that is labeled in a subsequent process by an anti-antibody or another marker molecule linked to a label molecule, such as an HRP, or AP (Horse raddish peroxidase, or alkaline phosphatase).
  • the sample is assayed using an appropriate method for the label used, such as by photometric analysis, e.g. a colorimetric assay where the label is an enzyme, or by fluorescence where the label is a fluorochrome, or by a combination of these techniques.
  • photometric analysis e.g. a colorimetric assay where the label is an enzyme, or by fluorescence where the label is a fluorochrome, or by a combination of these techniques.
  • Marker molecules are molecules or complexes of molecules that bind to other molecules. Marker molecules thus may bind to molecules on entities, including the desired entities as well as undesired entities. Labeling molecules are molecules that may be detected in a certain analysis, i.e. the labeling molecules provide a signal detectable by the used method. Marker molecules, linked to labeling molecules, constitute detection molecules. Sometimes a marker molecule in itself provides a detectable signal, wherefore attachment to a labeling molecule is not necessary.
  • Marker molecules are typically antibodies, directed against human blood cells, or other antigens as exemplified in FIG. 1 , however may be directed against antigens from any organisms, e.g. mouse, yeast, or bacteria. Antibodies may be monoclonal or polyclonal and derived from any species e.g. man, mouse, rabbit, pig. The antigens are most often proteins but may also be other molecules, e.g. sugars, DNA, RNA, or small organic molecules.
  • marker molecules maybe, aptamers, proteins, peptides, small organic molecules, natural compounds (e.g. steroids), non-peptide polymers, MHC multimers (including MHC-dextramers, MHC-tetramers, MHC-pentamers and other MHC-multimers), or any other molecules that specifically and efficiently bind to other molecules are also marker molecules, as exemplified in Table 1.
  • marker molecules bind molecules associated with an entity inhere defined as the target for the marker molecule.
  • marker molecules examples include marker molecules and corresponding target molecules. Examples of marker molecules and corresponding target molecules are given below, and in FIG. 1 and Table 1.
  • Table 1 Example, Marker Molecules and Description of Example Targets, and Example Diagnostic Value.
  • the table List lists examples of marker molecules, being antibodies or other molecules, what the marker binds, what sort of molecule the marker is and the value of different marker groups is, which may be used in contra or pro selection in an identification assay. Thus may be used as pro or contra selective assay's as described in this invention.
  • Her2 FISH probe aptamer NEU, or HER2 Brest cancer cell gene DNA amplification TOP2A FISH probe aptamer TOP2A (DNA) proliferating cells e.g. malignant cells, cancer cells gene amplification anti-CD8 antibodies
  • Antibody/protein CD4 T cell receptor T-helper cells Phosphor tyrosine Antibody/protein Tyrosine activated cells, e.g. specific antibody phosphorylated cancer cells proteins
  • Phosphor Serine Antibody/protein Serine activated cells e.g.
  • Estrogen Small organic molecule ER (estrogen Cells, including Brest steroid receptor) cancer cells Morphine small organic molecule Opioid receptor neural cells MHC dextramer protein HLA-complex, T- Antigen specific T cell receptor cells EGF (epidermal Protein EGF receptor Brest cancer cells growth factor) Activated MEK Intracellular protein MAPKinase Growing cells kinase signaling molecule serine and or tyrosine kinase Calcium (Ca 2+ ) Ion Calcium binding Mammalian cells, e.g.
  • RAF protein e.g muscle cells calmodulin Anti-Phosphor serin, Phosphorylated Protein Activated cellular Cancer cells, growing threonine and signaling cells thyrosin, antibodies molecules Specific protein Peptide other proteins, e.g. Cells expressing binding domains, the RAF protein target protein, “growth such as RAS's RAF activated cells” binding domain.
  • Anti-BrdU antibodies Protein/antibody BrdU incorporated Cell growth analysis into cellular DNA DNA fluorescing Small organic molecules DNA Cells containing DNA dyes e.g.
  • Marker molecules may bind to the surface of an entity (e.g. membrane proteins of a cell), or may bind to the interior of the entity (e.g. molecular structures within a cell, such as proteins, membranes, or nucleic acids).
  • entity e.g. membrane proteins of a cell
  • interior of the entity e.g. molecular structures within a cell, such as proteins, membranes, or nucleic acids.
  • Labeling molecules are molecules that are detectable in a specific assay, and the amount of labeling molecule may be quantified.
  • Radioactive radiation radioactive radiation
  • absorption of light e.g. dyes, chromophores
  • emission of light after excitation fluorochromes
  • nuclear magnetic resonance form paramagnetic molecules NMR
  • reflection of light scatter from e.g. such as gold-, plastic- or glass-beads/particles of various sizes and or shapes.
  • labelling molecules may have an enzymatic activity, by which it catalyze a reaction between chemicals in the near environment of the labeling molecules, producing a signal which include production of light (chemi-luminescence) or precipitation of chromophors, dyes, or a precipitate that can be detected by an additional layer of detection molecules.
  • the enzymatic product may deposit at the location of the enzyme, in cell based assays, react with the membrane of the cell, or diffuse into the cell to which the enzymatic marker is attached, in this way labeling the cell.
  • a single labelling molecule on a marker does not always generate sufficient signal intensity.
  • the signal intensity may be improved by assembling single label molecules into large multi-labelling compounds, containing two or more label molecule residues.
  • Multi-label compounds may be achieved by covalent or non covalent, association of labelling molecules with a major structural molecule.
  • Such structures may be synthetic or natural polymers e.g. dextrans, DNA, PNA, a protein such as streptavidin.
  • the label molecules in a multi-labelling compound may all be of the same type or may be a mix of different labelling molecules.
  • the table lists labelling substances that may be used as labeling molecules in any of the described assays. Also is described how the label “gives the signal”, and the physical appearance of the signal, as well as the detection principle employed, may any of these labels be used in the herein described methods.
  • the habten can be recognized by a detection molecule.
  • Example fluorochromes Example fluorochromes which may be used to label any of the marker molecules described in this invention, and the maximum excitation and the emission wavelengths for these fluorochromes.
  • Table 3b Example Preferable Fluorochrome Families.
  • Fluorochrome family Example fluorochrome AlexaFluor ®(AF) AF ® 350, AF405, AF430, AF488, AF500, AF514, AF532, AF546, AF555, AF568, AF594, AF610, AF633, AF635, AF647, AF680, AF700, AF710, AF750, AF800 Quantum Dot (Qdot ®) based Qdot ® 525, Qdot ® 565, Qdot ® 585, Qdot ® 605, Qdot ® 655, dyes Qdot ® 705, Qdot ® 800 DyLight TM Dyes (DL) DL549, DL649, DL680, DL800 Small fluorescing dyes FITC, Pacific Blue TM, Pacific Orange TM, Cascade Yellow TM, Marina blue TM, DSred, DSred-2, 7-AAD
  • the labelling molecules may be associated, linked or covalent bound to the marker molecule and added to the sample.
  • the marker molecule are first allowed to associate with the specific target, then the label molecule is added, binding specifically to the marker molecule.
  • the latter involves that the marker and the label is modified in a way that will allow a specific association between the marker and the label. This may be archived by any means that mediates a specific interaction between the marker and the labelling molecule.
  • Means as described for the association between the maker molecule and the target may be applied to mediated specific binding pairs of molecules, such as Antigen/Antibodies, streptavidine/biotin, nucleotides base pairing (DNA:DNA, RNA:RNA, RNA:DNA), or PNA with PNA, PNA with DNA or RNA, where the bead is attached to one of the partner molecules (e.g. the antigen) and the label is attached to the other partner molecule (e.g. the antibody).
  • the partner molecules e.g. the antigen
  • the label is attached to the other partner molecule (e.g. the antibody).
  • the typical label is detected by its fluorescence, examples are given in Table 3.
  • a positive detection is based on the presents of light from a single fluorochrome, but in other techniques the signal is detected by a shift in wavelength of emitted light; as in FRET based techniques, where the exited fluorochrome transfer its energy to an adjacent bound fluorochrome (and marker) that emits light, or when using Ca 2+ chelating fluorescent probes, which change the emission (and absorption) spectra when binding to calcium.
  • fluorochrome or fluorochrome combinations on a specific marker molecules in particular antibodies, or MHC'multimers are more or less unlimited, a few rules may be employed to give better resolution and clearer identification of the specific cell population that are assayed.
  • labeling principles preferably used and developed for use in stationary cytometry, but may be used in other detection principles as well, such as flowcytometry.
  • the grouping describes the labels, name, what the effector molecule is, how it manifest itself in the assay, any secondary binding pair if used in a second layer of detection.
  • the sample can be either solid or a fluid suspension. If the sample is fluid it may be applied to the analytical instrument by positive or negative pressure applied to the sample. In flowcytometry, the sample is a suspension of entities, which are moved to and centered in the flow cell (interrogation point) by co-flow with sheath fluid, or is directly injected into the instrument. If the sample is a solid it is typically placed in the instrument in the form of a thin slide of material on a glass plate. Solid materials may be processed physical and/or enzymatically into a suspension, or assayed directly without further manipulation.
  • analysis shall involve the use of marker molecules that associate with specific entities, and may also involve inherent physical characteristics measured by the instrument employed e.g. scatter parameters in flowcytometry. By these parameters the detection of specific entities are allowed.
  • selection also termed enrichment, isolation or sorting shall involve the use of marker molecules that associate with certain entities, and may as well involve inherent physical characteristics that can be detected by the technique employed.
  • Example entities are cells and cell like particles, micelles and liposome's, polymer beads, and supramolecular structures, or any other molecular complex or particle or sub-component, detectable or selectable by the use of marker molecules, and specific inherent characteristics.
  • Cytometry can in principle be divided into two different categories: Cytometry and batch-based analysis. Cytometry may be further divided into flow cytometry and stationary cytometry. The following introduces analytical principles applied in the various analysis.
  • Cytometry allows detection and investigation of physical and/or chemical properties of each entity in the sample analyzed. Cytometry may be grouped into flow cytometry (e.g., fluorescence-activated cell sorting (FACS)) and stationary cytometry (e.g. immunohistochemistry (IHC)). In flow cytometry the sample is in a liquid form, a suspension of entities which is flowing through an interrogation-point, whereas, in stationary cytometry the sample is typically a solid.
  • flow cytometry e.g., fluorescence-activated cell sorting (FACS)
  • stationary cytometry e.g. immunohistochemistry
  • Flow cytometry allows detection of a single entity with a specified set of characteristics, within a population of entities with other sets of characteristics.
  • a major advantage of flowcytometry is that it allows rapid analysis of multiple parameters for each individual entity, simultaneously.
  • the sample is transported to the flow cell by applying pressure to the sample tube and focused in the flow cell using sheath fluid.
  • a sheath fluidic system accelerates each entity into the flow-cell resulting in a focused line of entities. As a result, the entities pass through the interrogation point one at a time.
  • micro-fluidics-based, flow-on-a-chip system a “dry” flow cytometer
  • the sample is passed through the interrogation point by the means of micrometer-scale tubing's, using capillary forces acting on the liquid in a capillary tube.
  • Labelling molecules can be molecules that absorp, excite, or modify radiation, e.g light, and radioactivity. Obviously, the detection methods reflect this.
  • the most commonly used detection parameters are the scatter and fluorescence parameters, but other means of detection, e.g. color (absorption) and radioactivity may be used as well.
  • the scatter parameters are defined as the quantity of light detected in a forward (FSC) and a sideward or 90° scatter (SSC), or in any other defined angle to the light source and the sample stream. These measurements provide information on characteristics such as relative size, transparency and granularity of the entity.
  • the fluorescence parameters describe the fluorescence that is emitted from the detection molecules associated with the entity.
  • One or more lasers of well defined wavelengths are used to generate scatter light from, and to excite the fluorochromes associated with, each entity.
  • a common combination of lasers is the 405 nm-, 488 nm- and 635 nm-lasers, as used in the CyAn ADPTM flowcytometer.
  • the number of lasers may be more or less than that.
  • lasers emitting tuneable and multiple wavelengths of light conventional light sources or diode-based light sources can also be employed. Additionally, one may measure transmitted (absorbed), or reflected light, rather than fluorescence, from the entities.
  • Stationary cytometry is primarily employed in analysis of solid samples (e.g. slices of paraffin embedded tissue or slices of frozen tissue), however also smears of liquid samples may be analyzed by this method. Probably the most common type of stationary cytometry is immunohistochemistry (IHC).
  • IHC immunohistochemistry
  • the assay technique employed in stationary cytometry typically involves immobilization of the tissue slice on a glass slice, carrying the sample through the assay steps to the final analysis.
  • the sample is “stained” or labeled, using principles as described for markers and labeling elsewhere herein, and in the following.
  • the means of detection typically involve photometric methods, microcopy and/or digital scanning of the stained sample. It may be simple light or fluorescence microscopy, for determination of chemiluminescence, morphology, shape, and fluorescence. Also, laser scanning techniques may be employed, where confocal microscopy or standard light microscopy is employed to give a 3- and 2-dimensional picture, respectively, of the sample. A digital image may be acquired, whereby the individual features e.g. light intensity at a given area of the sample can be determined.
  • batch-based techniques are here defined as methods that capture entities using marker molecules that bind to specific target molecules on the entity. Following the captured entities may be isolated and enriched.
  • the sample used in these assay is preferably liquid-based samples.
  • Liquid-based samples here means samples that by nature is liquid (e.g. blood, lymph) or samples that may be liquefied prior to analysis (e.g. suspensions of solid tissue, cultured cells in suspension, beads in suspension ect.).
  • a solid support may be any which is suited for immobilization, separation etc.
  • Non-limiting examples include particles, beads, biodegradable particles, sheets, gels, filters, membranes (e. g. nylon membranes), fibres, capillaries, needles, microtitre strips, tubes, plates or wells, combs, pipette tips, micro arrays, chips, slides, or indeed any solid surface material.
  • the solid or semi-solid support may be labelled, if this is desired.
  • the support may also have scattering properties or sizes, which enable discrimination among supports of the same nature, e.g. particles of different sizes or scattering properties, colour or intensities.
  • the sample is incubated with the solid support. After incubation the solid support is washed to remove unbound sample, and the entities that remain associated with the surface of the solid support are recovered. The sample is thereby enriched for entities defined by the specific marker. Following recovery the selected entities may be expanded or their number determined. Determination of the amount of entities may also be made when the entities are still on the solid support.
  • Methods for capturing entities can also be used for analysis and/or isolation of substances or entities secreted from entities in a sample or produced in the sample during preparation of the sample.
  • the marker molecules may be immobilized directly to the solid support or may be immobilized through a linker molecule.
  • the linker molecule can be an antibody specific for the marker molecule, a peptide or a any other molecule able to bind the marker molecule.
  • the linker may be attached directly to the solid support, the linker may be attached to the solid support through another linker or the linker is embedded in a matrix, e.g. a sugar matrix.
  • the advantage of capture methods is that it is possible to assay large sample volumes for entities carrying target molecules specific for various markers. This is simply done by passing the sample over the solid support (e.g. a column containing beads) or by adding the solid support (e.g. beads) directly into the sample allowing the entities to bind to its specific immobilized marker molecules. If the solid support is e.g. beads and these are incubated with the sample in suspension the beads and thereby the bound entities may be isolated by centrifugation or filtration and analyzed by e.g. flowcytometry or any other method as described in this invention. Using solid support surfaces in macro size (e.g. a chip or a glass slice), the isolation is simply done by removing the support from the suspension of entities and analyzed by any method as described elsewhere herein.
  • the solid support e.g. a column containing beads
  • the solid support e.g. beads
  • the amount of entities associated with markers on a solid support may be analyzed while bound to the solid support or the entities may be released/recovered and analyzed or expanded and analyzed.
  • the identity and amount of captured entities may be assayed by methods involving staining with a second specific detection molecule, or by staining with a generic stain, e.g. protein, lipid, or DNA stain.
  • a generic stain e.g. protein, lipid, or DNA stain.
  • the target molecule exposed on the entity is defined by the bead, area or solid support to which it binds.
  • the captured entities may be characterized further by employing a secondary detection layer, consisting of marker molecules directed against other targets, to the captured entities.
  • this second detection layer may release bound entities from the solid support by competing with the immobilized marker molecules for binding to defined targets.
  • the analysis may be supplemented with inherent characteristics of the entity, i.e. characteristics that can be detected without the use of marker molecules. Examples of such characteristics include enzymatic activity in a cell, secretion of molecules from a cell, auto-fluorescence from the entity, granularity, size, morphology in general ect.
  • Fluorochromes may be detected by photometry such as a laser-based system e.g. flow cytometry or by fluorescence microscopy. Enzymatic precipitation of dye molecules or chemiluminescence's may be examined by photometry or standard light microscopy. Radio nuclides may be detected with a Geiger Muller tube like method, a phosphor-imager, by a method based on the radioactivity induced excitation of molecules in a material that can by “read off” by induced emission of light. Lanthanides may be detected by its fluorescence, phosphorescence or NMR signal.
  • step C a number of further method steps can be carried out.
  • optional method steps include e.g. data acquisition (step D1) and sorting (partitioning) of the entities (D2).
  • a data set for a population of entities is obtained (for analysis); or alternatively (for the cytometry sorting process), a data set for each entity is obtained, allowing its possible isolation, or alternatively (for the batch-based sorting process), a sub-population of entities are physically isolated by e.g. immobilization to a solid support, and may optionally be released enriching a specific population of entities.
  • the phenotype of the individual entity is determined by its characteristic combination of properties. These can be physical properties of the entity, such as morphology of a cell, determined by immunohistochemistry, forward- and side-scatter of a cell in a flow cytometry analysis, or these can be chemical properties of the entity, such as ability of a certain cell type to bind specific marker molecules, or the level of certain ions, such as Calcium measured by ionophores.
  • gating strategy shall be used as the term describing the strategy of choosing the physical (e.g. scatter) and chemical (e.g. fluorescence from a labeled marker molecule) parameters to be used for identification of specific entities.
  • the gating strategy is defined by the marker molecules which have been added during sampling or sample preparation, as well as by the physical properties measured during the analysis. Example gating strategies are described in Examples.
  • the gating strategy may involve a positive as well as a negative selection of sub-populations.
  • desired entities may be labeled with one or several labels, that then define the target entities, whereas undesired entities may be labeled with one or several labels, whereby the unwanted entities are identified and may be disregarded (for analysis), dumped or deselected in a sorting process.
  • a systematical positive and/or negative selection of populations of entities with similar properties will lead to identification and enrichment of the entities with the desired properties.
  • cytotoxic T-cells are defined by being a lymphocyte, having low scatter properties, having the ability to bind to the anti-CD8 markers among others and not expressing CD4.
  • the specificity of cytotoxic T cells may be determined by the ability to associate specifically with a class 1 MHC-multimer carrying a specific peptide.
  • the sample may be stained with markers specific for CD4 and CD8 and with one ore more specific MHC multimers, e.g. MHC dextramers.
  • the gating strategy may then be first to deselect data points/cells with high scatter, and ability to bind the anti-CD4 marker.
  • a positive selection for the ability to bind both to the anti-CD8 marker and the MHC dextramers is employed.
  • the parameters are chosen with consideration to amount and kind of target molecules present on the entity, as well as the degree of spectral overlap between the contacteded fluorochromes, and that the markers do not interfere with other markers bound to the entity.
  • the quality of a flow cytometry analysis depends significantly on the gating strategy chosen.
  • a gating strategy is applied that involves the labeling of more than one marker molecule with the same labeling molecule. This allows collective identification of more than one population of entities. If such populations are negatively selected, it has additional advantage, as these labels are not situated on the entity of interests, thus any potential interference between the detection molecules, such as spectral overlap, undesired FRET, quenching, or marker/label interference is eliminated between these.
  • Another preferred embodiment is therefore preferable to use many e.g. 5 negative selection marker molecules, e.g. labeled with the same labeling molecule and fewer positive selection markers, e.g. one, than using e.g. 6 positive selection markers and no negative selection markers.
  • the consequence of the first choice is that the cells of interest only has one marker bound, where as, in the latter, 6 markers are bound to the cell of interest.
  • markers that bind to undesired cells may all be labeled with the same label, e.g. fluorescein.
  • markers could be anti-CD4, anti-CD14, anti-CD15, anti-CD19, and anti-CD71 antibodies, all labeled with fluorescein (FITC), as these markers will bind to the undesired T-helper cells, monocytes, granulocytes, B-lymphocyte and red blood cells (RBC), respectively.
  • FITC fluorescein
  • the spillover from fluorescein into the RPE channel can be neglected as fluorescein and RPE will never be situated on the same entity.
  • the data obtained on the fluorescein labeled entities may then be disregarded or the cells may be dumped during a sorting process.
  • Identification and or analysis of a particular group or family of entities within a sample may require a number of different parameters, i.e. detections molecules, and labels, which may not be possible to detect on the instrument employed. Grouping of markers may help solved such problem.
  • CTL's Cytotoxic T Lymphocytes
  • Borealis a family of pathogens e.g. CMV, EBV, BK viruses or Borealis
  • MHC multimers each labeled with a different label, where each MHC multimer reagent carries a different peptide or a different allele or combinations thereof identifying individual antigen I specific T cell clones directed against different pathogen members of the pathogen family.
  • An alternatively approach is therefore to label all or subsets of the involved MHC multimer reagents with the same labeling molecule.
  • the advantage is that it reduces the total number of markers, freeing parameters for additional analysis of other characteristics of the specific CTL, such as the activation status.
  • Grouping of markers by using the same label on different markers is not restricted to grouping of the above mentioned markers but can be used on any group of markers. Identification of a group of entities by using the same labeling molecule on different markers may be used for positive as well as negative selection of different entities.
  • the primary sample contained 100 cells of the desired kind.
  • Particles may be detected and counted by e.g. their fluorescence or scatter characteristics in flow cytometry, or by visual inspection in microscopy.
  • the beads may be labeled internally, by absorption of e.g. fluorescent dyes (such beads are called “hard-dyed” beads), or fluorochromes may be chemically cross-linked, or adsorbed to the bead surface.
  • the fluorochrome can also be attached by specific binding pairs of molecules, such as Antigen/Antibodies, streptavidine/biotin, nucleotides base pairing (DNA:DNA, RNA:RNA, RNA:DNA), or PNA with PNA, PNA with DNA or RNA, where the bead is attached to one of the partner molecules (e.g. the antigen) and the label is attached to the other partner molecule (e.g. the antibody).
  • the relative amount of certain specific entities is desired, relative to other entities in the sample.
  • the amount of blood plates is often relatively constant in different individuals, and therefore, the number of specific cells in a blood sample may be normalized with the measured amount of blood plates in the sample.
  • the absolute count of a subpopulation of entities is desirable, such as the total amount of CD4 positive cells, or specific MHC-multimer positive cells in the blood sample of a patient.
  • the spectra may overlap, resulting in an error on the intensity measured in a specific wavelength interval, by light from other fluorochromes.
  • this “spillover” of light may be compensated for.
  • the spillover coefficients between detectors measuring in different wavelengths intervals are determined mathematically.
  • the spillover coefficient can be manually calculated, or can be calculated by automated software, such as SummitTM software.
  • the spillover coefficients are used to correct the measured signal intensity for each fluorochrome in multicolor flow cytometry for spillover from the other fluorochromes. This may be performed by employing linear algebra on the intensity measured for each entity, subtracting the signals coming from the other fluorochromes from that of the primary fluorochrome for that detector, when displaying the results.
  • digital deconvolution can help distinguish different features (e.g. colors, and morphologies) of the sample.
  • the primary goal is to define the localization of the marker molecule, and only roughly determinate the amount of marker bound defined by the label used.
  • differentiation between the labeling molecules used e.g. the fluorochromes in fluorescence microscopy
  • each marker may be performed by making overlays of exposures done with different emission filters, thus if digitalized the spillover correction can be done in the same way as described for flow cytometry.
  • a high resolved digitalized color image obtained be e.g. CCD's may contain more information about the spectral properties of the emitted light (higher resolution of the wavelength data recorded), as well as much higher amount of light recorded, then does a PMT detection in flow cytometry, therefore, by knowing the spectral properties of each of the fluorochromes it is possible to deconvolute the intensity from each of the individual fluorochromes, using mathematical overlay of their spectra's.
  • Analysis and sorting depend on the same data. In an analysis of a sample, data may be acquired for all the entities of the sample first, where after the data can be analyzed when the experiment is over. In sorting, this is not possible.
  • a gating strategy is set up based on a small sample acquired.
  • sort strategy/gating strategy defining a set of criteria for which each entity is analyzed. Based on this analysis in real time, a sort decision is taken; either isolate (sort out) or deselect, dumped, the entity.
  • the flow sorter, MoFlo (Dako NS) can administrate positive sorting into four different population of entities, and dumping (or collecting) the rest.
  • gating criteria that might be used to select (sort for) CTLs of a given specificity.
  • the cell bind to the following marker molecules: MHC-dextramer with a particular specificity, anti-CD3 defining lymphocyte cells, anti-CD8 defining the CTL of which the antigen specific MHC-dex positive cells will be found, but does not bind the following marker molecules: anti-CD15, and anti CD4, which are used for counter selection of granulocytes, and T-helper cells as well as monocytes, respectively.
  • the anti-CD4 and anti-CD15 antibodies is used as dump gate, and the MHC dextramer, anti-CD8 and anti-CD3 as positive selecting markers.
  • Automatic population tracking software such as CyTrackTM, may be employed during the sorting process to compensate for such drifts.
  • the software will change the sort gate so that it always fits to the original population identified in the sample.
  • the software is tracking changes in several dimensions, where each dimension is a parameter that is measured and recorded (e.g. fluorescence at a certain wavelength interval, and forward and side scatter).
  • the choice of marker molecules is critical to obtain the best possible result.
  • the assay strategy (defined here as the combination of sample, marker and labeling molecules used in the gating strategy, and the analytical instruments employed in a particular analysis) and the choice of other reagents must be properly coordinated to achieve the optimal result.
  • the reagents must be constructed and combined in a way so as to maximize the information output, by e.g. optimizing the label choice for each particular marker molecule, relative to the other reagents, and methods used.
  • the number and kind of markers e.g. 10 marker reagents, A-J
  • the full analysis for a process involving e.g. 10 marker reagents, A-J can thus be described as follows:
  • Each of the (labeled) markers A-J may be chosen from, but not restricted to the list of markers and labels exemplified in FIG. 1 and table 1 to 3, and within this invention.
  • the task likewise may be one of the tasks described above (e.g. sorting among different entities by flow cytometry, detection of particular cell morphologies by an immunohistochemical assay from a fixed paraffin embedded tissue sample, or enrichment of particular blood cells by adsorption to beads). If the total number of combinations of markers and labels is n, and the total number of tasks is m, then the different processes involving 10 labeled markers are:
  • Assembly 1 Identification of a specific cell population in a sample.
  • A A detection molecule specifically binding, and identifying a cell population of interest.
  • Z Flow cytometric analysis of the sample on any flow cytometer capable of detecting the label employed.
  • Assembly 2 Identification of a specific cell population in a sample.
  • A A detection molecule specifically binding, and identifying a cell population of interest.
  • Z Flow cytometric analysis of the sample on any flow cytometer capable of measuring scatter parameters and the labels employed.
  • Assembly 3 Identification of a specific cell population in a sample.
  • A A detection molecule specifically binding, and identifying a cell population of interest.
  • B A detection molecule specifically binding to any cell population different to the cell population of interest.
  • Z Flow cytometric analysis of the sample on any flow cytometer capable of measuring scatter parameters and the labels employed.
  • A A labeled MHC-dextramer, carrying a peptide identical to a peptide sequence in a specific protein, of interest.
  • B A detection molecule specifically binding, and identifying CTL's, where the label is different form that used on the MHC-dextramer.
  • Z Flow cytometric analysis of the sample on any flow cytometer capable of detecting the label employed.
  • A A labeled MHC-dextramer, carrying a peptide identical to a peptide sequence in a specific protein, of interest.
  • B A detection molecule specifically binding, and identifying CTL's, labeled with a labeling molecule different form that used on the MHC-dextramer.
  • C A detection molecule specifically not binding to CTL's, labeled differently form those used in A and B.
  • Z Flow cytometric analysis of the sample on any flow cytometer capable of measuring scatter parameters and the labels employed.
  • A A labeled MHC-dextramer, carrying a peptide identical to a peptide sequence in a specific protein, of interest.
  • B CD8 antibody labeled with a labeling molecule different form that used on the MHC-dextramer.
  • Z Flow cytometric analysis of the sample on any flow cytometer capable of measuring scatter parameters and the labels employed.
  • A A labeled MHC-dextramer, carrying a peptide identical to a peptide sequence in a specific protein, of interest.
  • B CD8 antibody labeled with a labeling molecule different form that used on the MHC-dextramer.
  • Z Flow cytometric analysis of the sample on any flow cytometer capable of measuring scatter parameters and the labels employed.
  • A A labeled MHC-dextramer, carrying a peptide identical to a peptide sequence in a specific protein, of interest.
  • B CD8 antibody labeled with a labeling molecule different form that used on the MHC-dextramer.
  • C One or more than one labeled antibodies identifying cell populations different from the CTL's, all labeled with the same labeling molecule, different from that used for the MHC-dextramer, and the CD8 antibody.
  • Z Flow cytometric analysis of the sample on any flow cytometer capable of measuring scatter parameters and the labels employed.
  • A A labeled MHC-dextramer, carrying a peptide identical to a peptide sequence in a specific protein, of interest.
  • B CD8 antibody labeled with a labeling molecule different form that used on the MHC-dextramer.
  • C CD3 antibody labeled with a labeling molecule different form that used on the
  • MHC-dextramer and the CD8 antibody.
  • Z Flow cytometric analysis of the sample on any flow cytometer capable of measuring scatter parameters and the labels employed.
  • A A labeled MHC-dextramer, carrying a peptide identical to a peptide sequence in a specific protein, of interest.
  • B CD8 antibody labeled with a labeling molecule different from that labeling the
  • C CD3 antibody labeled with a labeling molecule different form that used on the MHC-dextramer, and the CD8 antibody.
  • D One labeled antibody identifying cell populations different from the CTL's, labeled with a labeling molecule different from that used for the MHC-dextramer, the anti-CD8, and anti-CD3 markers.
  • Z Flow cytometric analysis of the sample on any flow cytometer capable of measuring scatter parameters and the labels employed.
  • A A labeled MHC-dextramer, carrying a peptide identical to a peptide sequence in a specific protein, of interest.
  • B CD8 antibody labeled with a labeling molecule different from that labeling the MHC-dextramer.
  • C CD3 antibody labeled with a labeling molecule different form that used on the MHC-dextramer, and the CD8 antibody.
  • D More than one labeled antibodies, all labeled with the same labeling molecule, different from that used for the MHC-dextramer, the anti-CD8, and anti-CD3 markers.
  • Z Flow cytometric analysis of the sample on any flow cytometer capable of measuring scatter parameters and the labels employed.
  • Assembly 12 Identification of specific CTL's (Cytotoxic T Lymphocytes) involved in the immune response against a specific protein.
  • A A FITC MHC-dextramer, carrying a peptide identical to a peptide sequence in a specific protein, of interest.
  • B A CD8 antibody labeled with a labeling molecule different from FITC.
  • C One or more than one labeled antibody, all labeled with the same labeling molecule, different from FITC, and the label used for the CD8 antibody
  • Z Flow cytometric analysis of the sample on any flow cytometer capable of measuring scatter parameters and the labels employed.
  • A A RPE MHC-dextramer, carrying a peptide identical to a peptide sequence in a specific protein, of interest.
  • B A CD8 antibody labeled with a labeling molecule different from RPE.
  • C One or more than one labeled antibody, labeled with the same labeling molecule, different from RPE, and the label used for the CD8 antibody.
  • Z Flow cytometric analysis of the sample on any flow cytometer capable of measuring scatter parameters and the labels employed.
  • Assembly 14 Identification of specific CTL's (Cytotoxic T Lymphocytes) involved in the immune response against a specific protein.
  • A A APC MHC-dextramer, carrying a peptide identical to a peptide sequence in a specific protein, of interest.
  • B A CD8 antibody labeled with a labeling molecule different from APC.
  • C One or more than one labeled antibody, labeled with the same labeling molecule, different from APC, and the label used for the CD8 antibody.
  • Z Flow cytometric analysis of the sample on any flow cytometer capable of measuring scatter parameters and the labels employed.
  • Assembly 15 Identification of specific CTL's (Cytotoxic T Lymphocytes) involved in the immune response against a specific protein.
  • A A FITC labeled MHC-dextramer, carrying a peptide identical to a peptide sequence in a specific protein, of interest.
  • C PB labeled CD3 antibody.
  • D RPE labeled antibodies identifying cell populations different from the CTL's.
  • Z Flow cytometric analysis of the sample on any flow cytometer capable of measuring scatter parameters and the labels employed.
  • A A FITC labeled MHC-dextramer, carrying a peptide identical to a peptide sequence in a specific protein, of interest.
  • C PB labeled CD3 antibody.
  • D RPE labeled antibodies directed against monocytes and granulocytes, respectively.
  • Z Flow cytometric analysis of the sample on any flow cytometer capable of measuring scatter parameters and the labels employed.
  • A A FITC labeled MHC-dextramer, carrying a peptide identical to a peptide sequence in a specific protein, of interest.
  • Z Flow cytometric analysis of the sample on any flow cytometer capable of measuring scatter parameters and the labels employed.
  • A A FITC labeled MHC-dextramer, carrying a peptide identical to a peptide sequence in a specific protein, of interest.
  • CD4 RPE labeled CD4, CD14, and CD15 antibodies
  • Z Flow cytometric analysis of the sample on any flow cytometer capable of measuring scatter parameters and the labels employed.
  • A A FITC labeled MHC-dextramer, carrying a peptide identical to a peptide sequence in a specific protein, of interest.
  • Z Flow cytometric analysis of the sample on any flow cytometer capable of measuring scatter parameters and the labels employed.
  • A A RPE labeled MHC-dextramer, carrying a peptide identical to a peptide sequence in a specific protein, of interest.
  • C PB labeled CD3 antibody.
  • D FITC labeled antibodies identifying cell populations different from the CTL's.
  • Z Flow cytometric analysis of the sample on any flow cytometer capable of measuring scatter parameters and the labels employed.
  • A A RPE labeled MHC-dextramer, carrying a peptide identical to a peptide sequence in a specific protein, of interest.
  • C PB labeled CD3 antibody.
  • D FITC labeled antibodies directed against monocytes and granulocytes, respectively.
  • Z Flow cytometric analysis of the sample on any flow cytometer capable of measuring scatter parameters and the labels employed.
  • A A RPE labeled MHC-dextramer, carrying a peptide identical to a peptide sequence in a specific protein, of interest.
  • Z Flow cytometric analysis of the sample on any flow cytometer capable of measuring scatter parameters and the labels employed.
  • Assembly 23 Identification of specific CTL's (Cytotoxic T Lymphocytes) involved in the immune response against a specific protein.
  • A A RPE labeled MHC-dextramer, carrying a peptide identical to a peptide sequence in a specific protein, of interest.
  • Z Flow cytometric analysis of the sample on any flow cytometer capable of measuring scatter parameters and the labels employed.
  • Assembly 24 Identification of specific CTL's (Cytotoxic T Lymphocytes) involved in the immune response against a specific protein.
  • A A RPE labeled MHC-dextramer, carrying a peptide identical to a peptide sequence in a specific protein, of interest.
  • Z Flow cytometric analysis of the sample on any flow cytometer capable of measuring scatter parameters and the labels employed.
  • Assembly 25 Identification of specific CTL's (Cytotoxic T Lymphocytes) involved in the immune response against a specific protein.
  • A A APC labeled MHC-dextramer, carrying a peptide identical to a peptide sequence in a specific protein, of interest.
  • C PB labeled CD3 antibody.
  • D FITC labeled antibodies identifying cell populations different from the CTL's.
  • Z Flow cytometric analysis of the sample on any flow cytometer capable of measuring scatter parameters and the labels employed.
  • Assembly 26 Identification of specific CTL's (Cytotoxic T Lymphocytes) involved in the immune response against a specific protein.
  • A A APC labeled MHC-dextramer, carrying a peptide identical to a peptide sequence in a specific protein, of interest.
  • C PB labeled CD3 antibody.
  • D FITC labeled antibodies directed against monocytes and granulocytes, respectively.
  • Z Flow cytometric analysis of the sample on any flow cytometer capable of measuring scatter parameters and the labels employed.
  • A A APC labeled MHC-dextramer, carrying a peptide identical to a peptide sequence in a specific protein, of interest.
  • Z Flow cytometric analysis of the sample on any flow cytometer capable of measuring scatter parameters and the labels employed.
  • Assembly 28 Identification of specific CTL's (Cytotoxic T Lymphocytes) involved in the immune response against a specific protein.
  • A A APV labeled MHC-dextramer, carrying a peptide identical to a peptide sequence in a specific protein, of interest.
  • Z Flow cytometric analysis of the sample on any flow cytometer capable of measuring scatter parameters and the labels employed.
  • A A APC labeled MHC-dextramer, carrying a peptide identical to a peptide sequence in a specific protein, of interest.
  • Z Flow cytometric analysis of the sample on any flow cytometer capable of measuring scatter parameters and the labels employed.
  • Assembly 30 Identification of specific CTL's (Cytotoxic T Lymphocytes) involved in the immune response against a specific protein.
  • A A unlabeled MHC-dextramer, carrying a peptide identical to a peptide sequence in a specific protein, of interest, and exposing a hapeten, a binding site for a specific detection molecule, e.g. the binding site on streptavidine for biotin.
  • B A detection molecule specific for the hapten on the MHC-dextramer, such as fluorochromes linked to Biotin.
  • B A CD8 antibody labeled with a labeling molecule different from that used in “B”.
  • C One or more than one labeled antibody, all labeled with the same labeling molecule, different from the one used in “B”, and the label used for the CD8 antibody.
  • Z Flow cytometric analysis of the sample on any flow cytometer capable of measuring scatter parameters and the labels employed.
  • Assembly 31 Identification of specific CTL's (Cytotoxic T Lymphocytes) involved in the immune response against a specific protein.
  • A A unlabeled MHC-dextramer, carrying a peptide identical to a peptide sequence in a specific protein, of interest, and having free binding sites for Biotin
  • B Multiple fluorochromes, e.g fluorescein residues linked together and linked to a biotin molecule.
  • D RPE labeled antibodies identifying cell populations different from the CTL's.
  • Z Flow cytometric analysis of the sample on any flow cytometer capable of measuring scatter parameters and the labels employed.
  • A A unlabeled MHC-dextramer, carrying a peptide identical to a peptide sequence in a specific protein of interest, and having free binding sites for Biotin.
  • B Multiple fluorochromes, e.g fluorescein residues linked together and linked to a biotin molecule.
  • D RPE labeled CD4, and CD19 antibodies.
  • Z Flow cytometric analysis of the sample on any flow cytometer capable of measuring scatter parameters and the labels employed.
  • Assembly 33 Identification of specific CTL's (Cytotoxic T Lymphocytes) involved in the immune response against a specific protein.
  • A APC labeled MHC-dextramer, carrying a peptide identical to a peptide sequence in a specific protein of interest, labled with FITC.
  • C FITC labeled CD4, and CD19 antibodies.
  • Z Flow cytometric analysis of the sample on any flow cytometer capable of measuring scatter parameters and the labels employed.
  • Assembly 34 Identification of specific CTL's (Cytotoxic T Lymphocytes) and evaluate its activation status.
  • A APC labeled MHC-dextramer, carrying a peptide identical to a peptide sequence in a specific protein of interest, labled with FITC.
  • C FITC labeled CD4, and CD19 antibodies.
  • E Labeled CD45RA antibody carrying a label different for the one used in A-D
  • F Labeled CD25 antibody carrying a label different for the one used in A-E
  • H Labeled CD28 antibody carrying a label different for the one used in A-G
  • Z Flow cytometric analysis of the sample on any flow cytometer capable of measuring scatter parameters and the labels employed.
  • Assembly 35 Identification of a group of entities using the same labeling molecules on different markers defining these entities.
  • A Multiple marker molecules, all labeled with the same label molecule used for either positive or negative selection of entities.
  • Z Flow cytometric analysis of the sample on any flow cytometer capable of detecting the label employed.
  • Assembly 36 Identification of a group of entities using the same labeling molecules on different markers defining these entities.
  • A Multiple marker molecules, labeled with the same label molecule used for either positive or negative selection of entities of interest.
  • B Multiple marker molecules, labeled with the same label molecule different from those used in “A”, used for either positive or negative selection of entities of interest.
  • Z Flow cytometric analysis of the sample on any flow cytometer capable of detecting the label employed.
  • Assembly 37 Identification of a heterogeneous cell population in a sample.
  • A Multiple marker molecules molecule specifically binding any of the specific cells in the cell population of interest.
  • B A detection molecule specifically binding to any cell population different to the cell population of interest.
  • Z Flow cytometric analysis of the sample on any flow cytometer capable of measuring scatter parameters and the labels employed.
  • Assembly 38 Identification of a cell population in a sample.
  • A Multiple of marker molecules specifically binding any of the specific cells in the cell population of interest, labeled with the same label.
  • B A number of marker molecules different from those used in “A” specifically binding any population of cells different from cells in “A”, labeled with the same label.
  • Z Flow cytometric analysis of the sample on any flow cytometer capable of measuring the labels employed.
  • A Multiple MHC-dextramer reagents (more than one), each carrying a peptide homologues to a peptide sequence in the specific protein or proteins of interest, labeled with the same label.
  • Z Flow cytometric analysis of the sample on any flow cytometer capable of measuring scatter parameters and the labels employed.
  • Assembly 40 Identification of specific CTL's (Cytotoxic T Lymphocytes) involved in the immune response against a specific protein or proteins.
  • A Multiple MHC-dextramer reagents (more than one), each carrying a peptide homologues to a peptide sequence in the specific protein or proteins of interest, labeled with the same label.
  • B A number of marker molecules different from those used in “A” specifically binding any population of cells different from cells defined by the markers used in “A”, labeled with the same label different form the label that labeling the MHC-dextramers.
  • C Scatter parameters may be used in positive and or negative selection of cell populations.
  • Z Flow cytometric analysis of the sample on any flow cytometer capable of measuring scatter parameters and the labels employed.
  • Assembly 41 Identification of specific CTL's (Cytotoxic T Lymphocytes) involved in the immune response against a specific protein or proteins.
  • A Multiple MHC-dextramer reagents (more than one), each carrying a peptide homologues to a peptide sequence in the specific protein or proteins, of interest, labeled with the same label.
  • B One or more than one labeled antibody, labeled with the same labeling molecule, different from that used for the MHC-dextramer, e.g. anti-CD8, and anti-CD3 markers.
  • Z Flow cytometric analysis of the sample on any flow cytometer capable of measuring scatter parameters and the labels employed.
  • A Multiple MHC-dextramer reagents (more than one), each carrying a peptide homologues to a peptide sequence in the specific protein or proteins, of interest, labeled with the same label.
  • B CD8 antibody labeled with a labeling molecule different from that labeling the MHC-dextramer.
  • C Optional a CD3 antibody labeled with a labeling molecule different form that used on the MHC-dextramer, and the CD8 antibody.
  • D One or more than one labeled antibody, labeled with the same labeling molecule, different from that used for the MHC-dextramer, the anti-CD8, and the anti-CD3 markers, that are not recognizing the population of cells positive for the MHC reagent.
  • Assembly 43 Identification of specific CTL's (Cytotoxic T Lymphocytes) involved in the immune response against a specific group of organisms, or antigens e.g. CMV, EBV, or BK family of viruses, or Tumor antigens.
  • CTL's Cytotoxic T Lymphocytes
  • antigens e.g. CMV, EBV, or BK family of viruses, or Tumor antigens.
  • A Multiple MHC-dextramer reagents (more than one) labeled with the same label, each carrying a peptide homologues to a peptide sequence in proteins derived from group of organisms or tumor cells.
  • B A marker molecule labeled with a label different form that used in A that are used to select of deselect entities in the sample analyzed.
  • Z Flow cytometric analysis of the sample on any flow cytometer capable of measuring scatter parameters and the labels employed.
  • Assembly 44 Identification of specific CTL's (Cytotoxic T Lymphocytes) involved in the immune response against a specific group of organisms, or antigens e.g. CMV, EBV, or BK family of viruses, or Tumor antigens.
  • CTL's Cytotoxic T Lymphocytes
  • antigens e.g. CMV, EBV, or BK family of viruses, or Tumor antigens.
  • A Multiple MHC-dextramer reagents (more than one) labeled with the same label, each carrying a peptide homologues to a peptide sequence in proteins derived from members of the family of organism or tumor cells.
  • B CD8 antibody labeled with a labeling molecule different form that used on the MHC-dextramer.
  • C One or more than one labeled antibodies identifying cell populations either different from the CTL's, all labeled with the same labeling molecule, different from that used for the MHC-dextramer, and the CD8 antibody.
  • Z Flow cytometric analysis of the sample on any flow cytometer capable of measuring scatter parameters and the labels employed.
  • Assembly 45 Identification and activation analysis of specific CTL's (Cytotoxic T Lymphocytes) involved in the immune response against a pathogen family.
  • A Multiple MHC-dextramer reagents (more than one) labeled with the same label, each carrying a peptide homologues to peptide sequences in proteins derived from members of the pathogen family.
  • B CD8 antibody labeled with a labeling molecule different form that used on the MHC-dextramer.
  • C One or more than one labeled antibodies indicative for activation status of CTL's labeled with a labeling molecule different from that used for the MHC-dextramer, and the CD8 antibody.
  • Z Flow cytometric analysis of the sample on any flow cytometer capable of measuring scatter parameters and the labels employed.
  • the output of the analysis and sorting is analytical data and a population of entities, respectively.
  • the data may be evaluated to obtain a final conclusion, e.g. a diagnosis for a patient.
  • the cells may be further manipulated or analyzed; for example, the cells may be proliferated, matured and/or multiplied, for use in a therapeutic scheme for a patient.
  • the procedure may involve i) Examination of the acquired data by e.g. a physician, or software that performs an automated analysis of the data, to obtain a diagnosis for the patient.
  • the data, the conclusion reached, and the general patient information may be stored or tightly linked, ii)
  • the data may be stored centrally, in order to allow (secured) access from any computer on the www, enabling the data to be evaluated by individuals (e.g. a physician) not necessary present on the site where the data has been acquired, iii)
  • the data and diagnosis may be linked to the “LIS” (laboratorie information system) system of the hospital, by specialized software.
  • the sample may be phenotyped by cluster analysis of the data in n-dimensions, and compared to a reference, which typically will be a negative control as well as a statistical sample of healthy individuals. Evaluation of a statistical as well as a physical “healthy” sample enables the software to compare the value of the n parameters to the value typically obtained for healthy persons and patients, respectively, and/or compare to the cluster analysis of the healthy standard individual. Both approaches to data interpretaiton may form the basis of the diagnosis.
  • the enriched population of entities that have been obtained from the sorting process, or by batch-based isolation may be further manipulated, as described by the following examples, which refer to the case where certain cells have been selected in the sorting process:
  • steps Sampling (A)—Sample Preparation (B)—Assaying (C)—Data acquisition (D1)/Sorting (D2)—Interpretation of data (E)—Manipulating entities (F).
  • steps A, B and C are essential, steps D (D1 and D2); E and F are optional method steps.
  • n denote the totality of n different processes of a given category, including the choice of the sub-processes (embodiments of that particular method step) where the sub-process (embodiment) in question is not performed. n may be different for the individual sub-processes A, B, C, D, or E.
  • A1 A sampling process where the source is blood
  • A2 A sampling process where the source is semen
  • A3 A sampling process where the source is human Blood
  • A4 A sampling process where the source is human cerebrospinal liquid
  • A5 A sampling process where the source is animal blood
  • A6 A sampling process where the source is animal semen
  • A7 A sampling process where the source is blood collected in a vacutainer®, a closed test tube, where the blood is drawn by a vacuum in the tube.
  • A8 A sampling process where the source is human blood collected in a vacutainer®, a closed test tube, where the blood is drawn by a vacuum in the tube.
  • A9 A sampling process where the source is animal blood collected in a vacutainer®, a closed test tube, where the blood is drawn by a vacuum in the tube.
  • A10 A sampling process where the source is blood collected in a capillary tube.
  • A11 A sampling process where the source is human blood collected in a capillary tube.
  • A12 A sampling process where the source is animal blood collected in a capillary tube
  • A13 A sampling process where the source is blood collected in a volumetric capillary tube, for a specific volume collection.
  • A14 A sampling process where the source is human blood collected in a volumetric capillary tube, for a specific volume collection.
  • A15 A sampling process where the source is animal blood collected in a volumetric capillary tube, for a specific volume collection.
  • A16 A sampling process where the source is human semen collected in a capillary tube.
  • A17 A sampling process where the source is animal semen collected in a capillary tube.
  • A18 A sampling process where the source is human semen collected in a volumetric capillary tube, for a specific volume collection.
  • A19 A sampling process where the source is animal semen collected in a volumetric capillary tube, for a specific volume collection.
  • A20 A sampling process where the source is blood collected in a vacutainer®, a closed test tube, where the blood is drawn by a vacuum in the tube, containing anticoagulating agent(s).
  • A21 A sampling process where the source is human blood collected in a vacutainer®, a closed test tube, where the blood is drawn by a vacuum in the tube, containing anticoagulating agent(s).
  • A22 A sampling process where the source is animal blood collected in a vacutainer®, a closed test tube, where the blood is drawn by a vacuum in the tube, containing anticoagulating agent(s).
  • A23 A sampling process where the source is blood collected in a capillary tube, containing anticoagulating agent(s).
  • A24 A sampling process where the source is human blood collected in a capillary tube, containing anticoagulating agent(s).
  • A25 A sampling process where the source is animal blood collected in a capillary tube, containing anticoagulating agent(s).
  • A26 A sampling process where the source is blood collected in a volumetric capillary tube, for a specific volume collection, containing anticoagulating agent(s).
  • A27 A sampling process where the source is human blood collected in a volumetric capillary tube, for a specific volume collection, containing anticoagulating agent(s).
  • A28 A sampling process where the source is animal blood collected in a volumetric capillary tube, for a specific volume collection, containing anticoagulating agent(s).
  • A29 A sampling process where the source is human semen collected in a capillary tube.
  • A30 A sampling process where the source is animal semen collected in a capillary tube.
  • A31 A sampling process where the source is human semen collected in a volumetric capillary tube, for a specific volume collection.
  • A32 A sampling process where the source is animal semen collected in a volumetric capillary tube, for a specific volume collection.
  • A33 A sampling process where the source is liquefied solid tissue suspension
  • A34 A sampling process where the source is human liquefied solid tissue suspension
  • A35 A sampling process where the source is animal liquefied solid tissue suspension
  • A36 A sampling process where the source is cultures of bacteria
  • A37 A sampling process where the source is cultures of yeast
  • A38 A sampling process where the source is fermenting cultures of bacteria, to follow the growth and composition of the culture.
  • A39 A sampling process where the source is fermenting cultures of yeast, to follow the growth and composition of the culture.
  • A40 A sampling process where the source is chemical reactions mixtures
  • A41 A sampling process where the source is a chemical reactions mixtures, collected in a tube or capillary tube containing particles that enables assaying a component in the reaction mixture.
  • A41 A sampling process where the source is a chemical reactions mixtures, collected in a tube or capillary tube containing chemical reagent or reagents that enables assaying a component, or components in the reaction mixture.
  • A42 A sampling process where the source is drinking water.
  • A43 A sampling process where the source is drinking water collected in a tube containing reagents for a specific assay of the water content.
  • A44 A sampling process where the source is sewage.
  • A45 A sampling process where the source is sewage collected in a tube containing reagents for a specific assay of the content.
  • A46 A sampling process where the source is soil.
  • A47 A sampling process where the source is soil collected and resuspended, in a tube containing a specific reagent or reagents that enables assaying a component, or components in the soil sample.
  • A48 A sampling process where the source is urine, collected in a tube or capillary tube
  • A49 A sampling process where the source is urine, collected in a tube or capillary tube, containing a specific reagent or reagents that enables assaying a component, or components in the sample.
  • A50 A sampling process where the source is urine samples in a volumetric tube or capillary tube, containing a specific reagent or reagents that enables assaying a component, or components in the sample.
  • A51 A sampling process where the source is human urine collected in a volumetric tube or capillary tube, containing a specific reagent or reagents that enable assaying a component, or components in the sample.
  • A52 A sampling process where the source is urine, collected in a tube or capillary tube, for analysis using a flowcytometry based instrument
  • A53 A sampling process where the source is human urine collected in a volumetric tube or capillary tube, containing a specific reagent or reagents that enables assaying a component, or components in the sample, for analysis using a flowcytometry based instrument.
  • A54 A sampling process where the source is blood collected in a vacutainer®, a closed test tube, where the blood is drawn by a vacuum in the tube, containing anticoagulating agent, as EDTA, or Citrate, and antioxidants.
  • anticoagulating agent as EDTA, or Citrate, and antioxidants.
  • A55 A sampling process where the source is human blood collected in a vacutainer®, a closed test tube, where the blood is drawn by a vacuum in the tube, containing anticoagulating agent, as EDTA, or Citrate, and antioxidants.
  • anticoagulating agent as EDTA, or Citrate, and antioxidants.
  • A56 A sampling process where the source is animal blood collected in a vacutainer®, a closed test tube, where the blood is drawn by a vacuum in the tube, containing anticoagulating agent, as EDTA, or Citrate, and antioxidants.
  • anticoagulating agent as EDTA, or Citrate, and antioxidants.
  • A57 A sampling process where the source is blood collected in a capillary tube, containing anticoagulating agent, as EDTA, Citrate or Heparins, and antioxidants.
  • anticoagulating agent as EDTA, Citrate or Heparins, and antioxidants.
  • A58 A sampling process where the source is human blood collected in a capillary tube, containing anticoagulating agent, as EDTA, Citrate or Heparins, and antioxidants.
  • anticoagulating agent as EDTA, Citrate or Heparins, and antioxidants.
  • A59 A sampling process where the source is animal blood collected in a capillary tube, containing anticoagulating agent, as EDTA, Citrate or Heparins, and antioxidants.
  • anticoagulating agent as EDTA, Citrate or Heparins, and antioxidants.
  • A60 A sampling process where the source is human semen collected in a tube, containing antifreeze—agents as DMSO or glyserol.
  • A61 A sampling process where the source is animal semen collected in a tube, containing antifreeze—agents as DMSO or glyserol.
  • A62 A sampling process where the source is human semen collected in a capillary tube, containing “antifreeze-agents” as DMSO or glycerol.
  • A63 A sampling process where the source is animal semen collected in a capillary tube, containing “antifreeze-agents” as DMSO or glycerol.
  • A64 A sampling process where the source is blood collected in a donor blood-bag, containing anticoagulating agent, as EDTA, Citrate or Heparins, and antioxidants.
  • anticoagulating agent as EDTA, Citrate or Heparins, and antioxidants.
  • A65 A sampling process where the source is blood collected in a disposal container, containing anticoagulating agent, as EDTA, Citrate or Heparins, and antioxidants.
  • anticoagulating agent as EDTA, Citrate or Heparins, and antioxidants.
  • A66 A sampling process where the source is blood collected in container, containing fixative.
  • A67 A sampling process where the sample is collected in a volumetric container, containing internal standards, such as counting beads.
  • A68 A sampling process where the sample is collected in a volumetric container, containing reagents for the further analysis.
  • A69 A sampling process where the sample is collected in a container that is coated with a marker molecule selectively binding a specific component or components of the sample, enriching for the unbound entities in the suspension.
  • A70 A sampling process where the source is a sample collected in a container that is labeled with a RIF based chip or otherwise labeled, enabling tracking of the source during the different step in the analysis procedure, as well as the treatment that sample has gone through.
  • A71:A sampling process where the source is blood collected in a vacutainer®, a closed test tube, where the blood is drawn by a vacuum in the tube, containing anticoagulating agent, as EDTA, or Citrate, and also cell separation medium e.g. comprised of a polyester gel and a density gradient liquid.
  • anticoagulating agent as EDTA, or Citrate
  • cell separation medium e.g. comprised of a polyester gel and a density gradient liquid.
  • A72 A sampling process where the source is human blood collected in a vacutainer®, a closed test tube, where the blood is drawn by a vacuum in the tube, containing anticoagulating agent, as EDTA, or Citrate, and antioxidants and also cell separation medium e.g. comprised of a polyester gel and a density gradient liquid
  • A73 A sampling process where the source is animal blood collected in a vacutainer®, a closed test tube, where the blood is drawn by a vacuum in the tube, containing anticoagulating agent, as EDTA, or Citrate, and also cell separation medium e.g. comprised of a polyester gel and a density gradient liquid.
  • anticoagulating agent as EDTA, or Citrate
  • cell separation medium e.g. comprised of a polyester gel and a density gradient liquid.
  • A74 A sampling process where the source is blood collected in a capillary tube, containing anticoagulating agent, as EDTA, Citrate or Heparins, and also cell separation medium e.g. comprised of a polyester gel and a density gradient liquid.
  • anticoagulating agent as EDTA, Citrate or Heparins
  • cell separation medium e.g. comprised of a polyester gel and a density gradient liquid.
  • A75 A sampling process where the source is human blood collected in a capillary tube, containing anticoagulating agent, as EDTA, Citrate or Heparins, and also cell separation medium e.g. comprised of a polyester gel and a density gradient liquid.
  • anticoagulating agent as EDTA, Citrate or Heparins
  • cell separation medium e.g. comprised of a polyester gel and a density gradient liquid.
  • A76 A sampling process where the source is animal blood collected in a capillary tube, containing anticoagulating agent, as EDTA, Citrate or Heparins, and also cell separation medium e.g. comprised of a polyester gel and a density gradient liquid.
  • anticoagulating agent as EDTA, Citrate or Heparins
  • cell separation medium e.g. comprised of a polyester gel and a density gradient liquid.
  • A77:A sampling process where the source is blood collected in a vacutainer®, a closed test tube, where the blood is drawn by a vacuum in the tube, containing anticoagulating agent, as EDTA, or Citrate, and antioxidants, and also cell separation medium e.g. comprised of a polyester gel and a density gradient liquid.
  • anticoagulating agent as EDTA, or Citrate
  • antioxidants also cell separation medium e.g. comprised of a polyester gel and a density gradient liquid.
  • A78 A sampling process where the source is human blood collected in a vacutainer®, a closed test tube, where the blood is drawn by a vacuum in the tube, containing anticoagulating agent, as EDTA, or Citrate, and antioxidants, and antioxidants and also cell separation medium e.g. comprised of a polyester gel and a density gradient liquid
  • A79 A sampling process where the source is animal blood collected in a vacutainer®, a closed test tube, where the blood is drawn by a vacuum in the tube, containing anticoagulating agent, as EDTA, or Citrate, and antioxidants, and also cell separation medium e.g. comprised of a polyester gel and a density gradient liquid.
  • anticoagulating agent as EDTA, or Citrate
  • antioxidants also cell separation medium e.g. comprised of a polyester gel and a density gradient liquid.
  • A80 A sampling process where the source is blood collected in a capillary tube, containing anticoagulating agent, as EDTA, Citrate or Heparins, and antioxidants, and also cell separation medium e.g. comprised of a polyester gel and a density gradient liquid.
  • anticoagulating agent as EDTA, Citrate or Heparins, and antioxidants
  • cell separation medium e.g. comprised of a polyester gel and a density gradient liquid.
  • A81 A sampling process where the source is human blood collected in a capillary tube, containing anticoagulating agent, as EDTA, Citrate or Heparins, and also cell separation medium e.g. comprised of a polyester gel and a density gradient liquid.
  • anticoagulating agent as EDTA, Citrate or Heparins
  • cell separation medium e.g. comprised of a polyester gel and a density gradient liquid.
  • a sampling process where the source is animal blood collected in a capillary tube, containing anticoagulating agent, as EDTA, Citrate or Heparins, and antioxidants, and also cell separation medium e.g. comprised of a polyester gel and a density gradient liquid.
  • anticoagulating agent as EDTA, Citrate or Heparins, and antioxidants
  • cell separation medium e.g. comprised of a polyester gel and a density gradient liquid.
  • B1 A sample preparation where the sample is added reagents that protect the sample against oxidation.
  • B2 A sample preparation where the sample is added reagents that stabilize the sample, and protect against cell lysis, such as fixatives and sugar.
  • B3 A sample preparation where the sample is added biocides or fixation to protect the operator form potentially contamination danger, such as viral or bacterial infections.
  • B4 A sample preparation where the sample is treated for selectively enrichement of a sub component of the sample.
  • B5 A sample preparation where the sample is treated with a chemical that lyse a specific subpopulation of cells in the sample, such as lysis of RBC in full blood using UtiLyseTM, EasyLyseTM, FACSLyseTM, or other ammonium containing solutions and/or by osmotic shock.
  • a chemical that lyse a specific subpopulation of cells in the sample such as lysis of RBC in full blood using UtiLyseTM, EasyLyseTM, FACSLyseTM, or other ammonium containing solutions and/or by osmotic shock.
  • B6 A sample preparation where the sample is treated with a chemical that precipitate a specific sup-population of the cells, such as antibody mediated aggregation, or association with beads having a specific marker molecule on the surface.
  • a chemical that precipitate a specific sup-population of the cells such as antibody mediated aggregation, or association with beads having a specific marker molecule on the surface.
  • B7 A sample preparation where the sample is fractionated by centrifugation in a density mediating substance, such as ficoll, sucrose or glycerol, separating different entity- populations by their density.
  • a density mediating substance such as ficoll, sucrose or glycerol
  • B8 A sample preparation where the sample is fractionated by centrifugation through a mesh membrane separating smaller from larger entities.
  • B9 A sample preparation where the sample is exposed to magnetized beads linked to a marker molecule that specifically bind a specific entity, selective retained in a magnetic field.
  • B10 A sample preparation where the sample is added fluorescent beads for internal standards during the analysis of the sample.
  • B11 A sample preparation where a specific volume of the sample is added a specific number of beads for quantitative counting of entities in the sample.
  • B12 A sample preparation where the sample is added specific marker molecules, such as antibodies for detection of specific entities within the sample.
  • B13 A sample preparation where the sample is added specific detections molecules allowing for phenotyping of the sample entities, by detecting the labelling on the detection molecules in an appropriate assay.
  • B14 A sample preparation where the sample is filtered to remove debris that potentially could interfere with the analysis method, such as flowcytometry where debris can block the sample probe.
  • B15 A sample preparation where the sample is added into a microtitreplate, or test tube containing immobilized detection molecules such as labeled antibodies in a matrix of sugars.
  • B16 A sample preparation where the sample is added reagents by an automated pippetting device and subsequently automatically applied to the flowcytometer or another analysis instrument.
  • B17 A sample preparation where the sample is added a particle capeable of association with the detections molecules as a procedure control.
  • B18 A sample preparation where solid tissue is mechanical and enzymatical processed to produce a cell suspension, a sample applicable to flowcytometry after incubation with specific detection molecules.
  • B19 A sample preparation where the source is solid parafin fixed tissue, sliced, placed on coverglass, and used as sample, prepared for assaying by de-parafination, antigen retrieval and subsequent incubated with detection molecules.
  • B20 A sample preparation where the source is frozen tissue, sliced, placed on a coverglass, fixed using alcohol and used as sample, assayed by incubated with detection molecules.
  • B21 A sample preparation where the source is a cell suspension, smeared onto a coverglass, fixed using alcohol and used as sample and assayed by incubated with detection molecules.
  • C2 Analysis of the prepared sample using Cytometry as assay procedure where the detection is based on the intensity of the fluorochromes on the marker molecules.
  • C3 Analysis of the prepared sample using Cytometry as assay procedure where the detection is based on the halflife, (fluorescence decay time), of the fluorochromes on the marker molecules.
  • C4 Analysis of the prepared sample using Cytometry as assay procedure where the detection is based on the total spectra of the fluorochrome, measured by multi-channel PMT or photo-responsive chip technology.
  • C6 Analysis of the prepared sample using Stationary Cytometry, where detection is based on the enzymatic generation of precipitated dye molecules, or light emission.
  • C7 Analysis of the prepared sample using Stationary Cytometry, where detection is based on fluorescence labeled marker molecules, detected using a digitalized picture of the labeled sample.
  • C10 Analysis of the prepared sample using immunohistochemestry (IHC), as assay procedure.
  • C13 Analysis of the prepared sample using batch-based assay, where a specific marker molecules are immobilized on a solid support and used to capture the specific population of entities associating with it.
  • C14 Analysis of the prepared sample using batch-based assay, where a specific marker molecules are immobilized on a particle and used to capture the specific population of entities associating with it, subsequent detected be a second detections molecule using flowcytometry as final assay.
  • C15 Analysis of the prepared sample using batch-based assay, where a specific marker molecules are immobilized on defined arrears of a surface and used to capture the specific population or populations of entities associating with them, detected with a generic cell dye.
  • C16 Analysis of the prepared sample using batch-based assay, where a specific marker molecules are immobilized on defined arrears of a surface and used to capture the specific population or populations of entities associating with these arrears, detected with a specific detection molecule.
  • C17 Analysis of the prepared sample using batch-based assay, where a specific marker molecules are immobilized on a solid support and used to capture the population of entities associating with it, detected using an intrinsic property of the cells, such as an enzymatic activity, or a colour reaction with a cellular component.
  • C18 Sorting of the prepared sample using Cytometry.
  • C19 Sorting of the prepared sample using Cytometry, where the detection is based on the intensity of the fluorochromes on the detection molecules.
  • C20 Sorting of the prepared sample using Cytometry, where the detection is based on the half-life (fluorescence decay time), of the fluorochromes on the marker molecules.
  • C21 Sorting of the prepared sample using Cytometry, where the detection is based on the total spectra of the fluorochrome, measured by multi-channel PMT or photo-responsive chip technology.
  • C22 Sorting of the prepared sample using sheath fluid based flowcytometry.
  • C23 Sorting of the prepared sample using “dry” (e.g. flow-on-a-chip) flowcytometry.
  • C24 Sorting of the prepared sample using capillary based flowcytometry.
  • C26 Sorting of the prepared sample using batch-based assay, where a specific marker molecules are immobilized on a solid support and used to capture the specific population of entities associating with it.
  • C27 Sorting of the prepared sample using batch-based assay, where a specific marker molecules are immobilized on a particle and used to capture the specific population of entities associating with it by centrifugation.
  • C28 Sorting of the prepared sample using batch-based assay, where a specific marker molecules are immobilized on magnetic particle used to capture the specific population of entities associating it, by means of a magnetic field.
  • D2 The data generated using flowcytometry, are analyzed for the presence of MHC-mer mediated and anti-CD8 mediated double fluorescing entities in the sample.
  • D3 The data generated using flowcytometry, are analyzed for the presence of MHC-mer mediated, anti-CD8 mediated and anti-CD3 mediated triple fluorescing entities in the sample.
  • D4 The data generated using flowcytometry, are analyzed for the presence of MHC-mer mediated, anti-CD8 mediated and anti-CD3 mediated triple fluorescing entities in the sample that are identified as a subpopulation of the anti-CD45 mediated fluorescing population.
  • D5 The data generated using flowcytometry are analyzed using a “dump” gate strategy in which the MHC-mer mediated fluorescing entities are defined by not being labeled with certain makers that are known to define population of entities in which the MHC-mer binding entities will not be found.
  • D6 The data generated using flowcytometry are analyzed for the presence of MHC-mer mediated fluorescing entities in the sample, among entities defined by not being labeled with CD4 fluorescing antibodies.
  • D7 The data generated using flowcytometry are analyzed for the presence of MHC-mer mediated fluorescing entities in the sample, among the entities defined by not being labeled with CD4, CD19, or CD15 fluorescing antibodies.
  • D8 The data generated using flowcytometry are analyzed for the presence of MHC-mer mediated fluorescing entities in the sample, among the entities defined by not being labeled with all or a subset of CD4, CD14, CD15 CD19, and CD71 fluorescing antibodies and staining that defined the dead cells in the sample.
  • D9 The data generated using flowcytometry are analyzed for the presence entities defined by binding to MHC-mer detected by FITC fluorescence.
  • D10 The data generated using flowcytometry are analyzed for the presence entities defined by binding to MHC-mer detected by RPE fluorescence.
  • D11 The data generated using flowcytometry are analyzed for the presence entities defined by binding to MHC-mer detected by APC fluorescence.
  • D12 The data generated using flowcytometry are analyzed for the presence entities defined by binding to MHC-mer detected by PB (pacific blue) fluorescence.
  • D13 The data generated using flowcytometry are analyzed for the presence of entities defined by binding to MHC-mer detected by e.g labels exmplified in Table 3a and b.
  • D14 The data generated using flowcytometry are analyzed for the concentration of entities defined by binding to MHC-mer detected by e.g labels exmplified in Table 3a and b relative to the amount of a counting bead such as CytoCountTM.
  • D15 The data generated using flowcytometry are analyzed for the present of a solid phase fluorescent bead or beads each associated with a specific MHC-mer molecule, which has captured an antigen specific T-cell detected by a fluorescent generic cell stain.
  • D16 The data generated using flowcytometry are analyzed for the present of a solid phase fluorescent bead or beads each associated with a specific MHC-mer molecule, which has captured an antigen specific T-cell detected by a fluorescent DNA stain, such as Draq5 or Propidium iodide.
  • a fluorescent DNA stain such as Draq5 or Propidium iodide.
  • D17 The data generated using flowcytometry are analyzed for the present of a solid phase fluorescent bead or beads each associated with a specific MHC-mer molecule, which has captured an antigen specific T-cell detected by a fluorescent detection molecule staining other characteristic of the specific T cell population, such as CD8.
  • D18 Identification of antigen specific T-cells by a solid phase surface assay, for the presence of cells in an area coated with a specific MHC-mer molecule, by staining the cells with a generic cell stain such a protein stain.
  • D19 Identification of antigen specific T-cells by a solid phase surface assay, for the presence of cells in an area coated with a specific MHC-mer molecules, by staining of bound cells using a fluorescent detection molecule detecting other characteristic of the specific T cell population, such as CD8.
  • E1 Based on the analysed data from flowcytometry, the amount of a specific entity is calculated relative to another defined entity in the sample.
  • E2 Based on the analysed data from flowcytometry, the amount of an antigen specific T-cell is calculated relative to another defined cell type in the sample.
  • E3 Based on the analysed data from flowcytometry, the amount of an antigen specific T-cells are calculated relative to the amount of CD8 positive cells in the sample.
  • E4 Based on the analysed data from volumetric flowcytometry, the amount of a specific entity is calculated relative to the volume of sample analysed, giving a concentration of the specific cell/ ⁇ l sample.
  • E5 Based on the analysed data from volumetric flowcytometry, the amount of an antigen specific T-cells is calculated relative to the volume of sample analysed, giving a concentration of the specific cell/ ⁇ l sample.
  • E6 Based on the analysed data from flowcytometry, the amount of a specific entity is calculated relative to a known amount of beads added to the sample, such as CytoCountTM, giving the concentration of the specific entity in the sample, also called “one platform counting”.
  • E7 Based on the analysed data from flowcytometry, the amount of an antigen specific T-cell is calculated relative to a known amount of beads added to the sample, such as CytoCountTM, giving the concentration of this antigen specific T-cell in the sample.
  • E8 Based on the analysed data from flowcytometry, the amount of an antigen specific T-cell is calculated relative to a known amount of a constant cell population in the sample, such as the blood plates, giving the concentration of this antigen specific T-cell in the sample.
  • E9 The sorted cells are expanded in-vitro, and used in further characterization studies.
  • E10 The sorted antigen specific T-cells are expanded in-vitro, and re-introduced into the patients blood stream for increased immunity towards the specific antigen.
  • E11 The sorted antigen specific T-cells are expanded in-vitro, and re-introduced into the patients blood stream to be used as autologous cancer therapy.
  • A (sampling): Use of vacutainer to collect 5 mL human blood.
  • sample preparation 100 ⁇ l of full blood is transferred to a tube containing MHC HLA-A*0201/APC loaded with the peptide “NLVPMVATV” from the CMV virus and incubated for 10′ at room temperature in the dark, where after the gating reagents anti-CD3/FITC, anti-CD8/PB are Presently preferred methods include but is not limited to the following:
  • the compensated data are analysed for the presence of cells that are triple stained with anti-CD3, anti-CD8 and MHC-mer reagents.
  • E (interpretation of data): The number of anti-CD8 (and anti-CD3) stained cells and that of anti-CD8, MHC-mer (and anti-CD3) stained cells are determined. The frequency of specific MHC-mer binding cells, or antigen specific T-cells towards the CMV virus specific peptide is calculated as “ # of MHC-mer cells/total # of CD8 positive cells”.
  • Process 2 Concentration of Specific T-Cell in Human Peripheral Blood.
  • A (sampling): Use of vacutainer to collect 5 mL human blood.
  • sample preparation 100 ⁇ l of full blood is transferred to a tube containing MHC HLA-A*0201/RPE, loaded with the peptide “NLVPMVATV” from the CMV virus, and exactly 100 ⁇ l CytoCountTM bead suspension, subsequently the sample is incubated for 10′ at room temperature in the dark, where after the gating reagents anti-CD3/FITC, anti-CD8/APC are added followed by additional incubation and subsequent lysis of the RBC using the reagent EasyLyseTM, thus a lyse no wash procedure. For compensation controls the same procedure are performed with a anti-CD3/fluor staining for each fluor used in the experiment, without the counting beads.
  • the compensated data from the MHC-mer stainings are analysed for the presence and number of cells that are triple stained with anti-CD3, anti-CD8 and MHC-mer reagents, and that of the CytoCount beads acquired.
  • E (interpretation of data): The number of anti-CD3, anti-CD8, and MHC-mer triple stained cells is determined, as well as the number of CytoCount bead. The concentration of the specific MHC-mer binding cells, or antigen specific T-cells towards the CMV virus specific peptide in the full blood sample is calculated;
  • C specific T-cell (Counted # SpecificTcell ⁇ Conc bead stock )/Counted # beads acquired , where the concentration of beads are app. 1000 beads/ ⁇ l stated on the vial, the volume of the CytoCountTM beads and the blood in the sample is the same.
  • A (sampling): Use of vacutainer to collect 5 mL human blood, transfer 100 ⁇ l of the blood to “matrix prepared reagents multititter plate”, containing the solidified reagents.
  • sample preparation The matrix prepared reagents wells or tubes contain the reagents, one tube contains MHC HLA-A*0201/APC, loaded with the peptide “NLVPMVATV” from the CMV virus, and the gating reagents anti-CD3/RPE, anti-CD8/Alexa700, and anti-CD45/PB, as well as the “dump” gate reagents anti-CD4, anti-CD14, anti-CD15, anti- anti-CD19, and anti-CD71 all labeled with FITC, and 1,00 ⁇ 10 5 CytoCountTM beads.
  • Some individual wells contain the compensation control reagents, anti-CD3/fluor staining for each fluor used in the experiment, without the counting beads.
  • the cells that are stained for FITC are gated away and the anti-CD45/PB positive cells are selected for further analysis and gating.
  • the anti-CD45/PB positive cells from the MHC-mer stainings are analysed for the presence and number of cells that are triple stained with anti-CD3, anti-CD8 and MHC-mer reagents.
  • the amount of CytoCount beads acquired is likewise determined for each sample.
  • E (interpretation of data): The number of anti-CD45, anti-CD3, anti-CD8, and MHC-mer triple stained cells are determined, as well as the number of CytoCount bead. The concentration of the specific MHC-mer binding cells, or antigen specific T-cells towards the CMV virus specific peptide in the full blood is calculated;
  • C specific T-cell Dilution factor of blood ⁇ (Counted # specific T cell ⁇ Conc bead stock )/Counted # beads acquired , where the concentration of beads are app. 1000 beads/ ⁇ l stated on the vial, and the dilutions factor in this experiment setup is 2.
  • A (sampling): Use of vacutainer to collect 5 mL human blood.
  • Example preparation 2.5 ml of full blood is transferred to a tube containing MHC-mer/APC loaded with a specific peptide from the protein of interest and incubated for 10 min at room temperature in the dark, where after the gating reagents anti-CD3/FITC, anti-CD8/PB are added followed by additional incubation and the subsequent lysis of the RBC using the reagent EasyLyse, followed by a centrifugation and resuspension of the cells, thus a Lyse wash procedure.
  • For compensation controls 100 ⁇ l blood are stained individually with anti-CD3/fluor for each fluor used in the experiment.
  • the compensated data are analysed for the presence of cells that are triple stained with anti-CD3, anti-CD8 and MHC-mer reagents, and the sort gate are placed accordingly.
  • the cells that are falling within the sortgate will be sorted into a cell tube containing growth medium and serum.
  • E entity enrichment: The sorted cells are directly assayed again using a CyAnTM ADPTM flow cytometer, and the purity of the sample are judghed. If the purity is not sufficient for the purpose of the cells, a additional round of sorting can be performed, identical to the first.
  • the sorted antigen specific T-cells can be enriched by growth in-vitro, and subsequent administrated to the patient potentially increasing the immunity towards the specific protein represented by the peptide.
  • Magnetic particles are coupled or associated with MHC multimer loaded with a specific peptide from a protein of interest.
  • the MHC multimer carrying magnetic particles are added into the donor blood bag, and incubated while mixing.
  • C/D assaying and sorting
  • the magnetic beads and associated cells is washed, and by removing the magnetic field, the beads and associated cells are freed and can be resuspended into a growth medium, or used for other purposes.
  • the cells associated with the beads can be tested in conventional flow Cytometry, as described in the former processes for identification control of the harvested cells.
  • E entity enrichment: The enriched antigen specific T-cells can be expanded by growth in-vitro, and subsequent administrated to the patient potentially increasing the immunity towards the specific protein represented by the peptide.
  • Tissue, fixed and embedded in paraffin are sliced in thin slices and placed on a glass slice.
  • sample preparation The paraffin block is sliced and thin slices are plased on glass slices. The sample is deparafinated, follow by a antigen retrival, and stained for DNA using DAPI and subsequent incubation with detection reagents, substrate is added and allowed to react.
  • the markers used are; a antigen specific MHCmer/HRP reagent (Horse Radish Peroxidase coubled), and DAPI stain for cell identification.
  • the presence of antigen specific T cells is detected by the presipitation of a dye due to the HRP enz.
  • D Data acquistion: The sample is analyzed manually by light microscopy, or by digital imaging.
  • E Interpretation of Data: The amount of antigen specific T cells are determined by the precipitated color, correlated to the DAPI stain for positive cell identification, either manually of by automated software working on the digital image.
  • A (sampling): Use of vacutainer or other container containing anti-coagulant to collect 4-10 ml human blood.
  • MHC dextramers are MHC-peptide complexes e.g. generated by in vitro refolding, coupled to dextran through covalent or non-covalent interactions.
  • the dextramer furthermore comprises APC coupled to the same dextran molecules through covalent or non-covalent interactions.
  • the MHC dextramers may all carry the same MHC I allele or carry different MHC I alleles.
  • Borrelia bacteria here include any strain of the species Borrelia Burgdorferi, Borrelia Garinii or Borrelia Afzelii.
  • Example Borrelia proteins include but is not limited to OspA, OspB, OspC, OspE, OspG, VIsE, FlaB, FIaA, BmpA, BmpB, BmpC, BmpD, HSP90, CRASP-1, CRASP-2, DbpA, DbpB, MaIQ, BapA, other outer surface Borrelia proteins, other intracellular proteins of Borrelia and plasmid encoded proteins from Borrelia bacteria.
  • the antigenic peptides are preferably 9′mers but can also be 8′mers, 10′ers or 11′mers.
  • the MHC dextramers are incubated for 10′ at room temperature in the dark, then gating reagents including anti-CD3/PE, anti-CD8/PB and anti-CD4/FITC are added and incubation continued for 20′.
  • RBC is lysed by addition of lysis reagents e.g. EasyLyse then the sample is centrifuged, supernatant removed. Cells are washed by resuspension in buffer followed by centrifugation. Supernatant is removed and cells resuspended in buffer.
  • fixative is added.
  • E (interpretation of data): The presence of cells stained with MHC dextramer, anti-CD3, anti-CD8 and not stained with anti-CD4 are T cells specific for Borrelia antigens and their frequency can optionally be determined as follows. The number of anti-CD8 and anti-CD3 stained cells and that of anti-CD8, MHC dextramer and anti-CD3 stained cells are determined. The frequency of specific MHC dextramer binding cells, or antigen specific T-cells towards Borrelia specific peptide is calculated as “ # of MHC dextramer positive, CD3+, CD8+cells/total # of CD3+,CD8 positive cells”.
  • A (sampling): Use of vacutainer or other container containing anti-coagulant to collect 4-10 ml human blood.
  • MHC dextramers are MHC-peptide complexes e.g. generated by in vitro refolding, coupled to dextran through covalent or non-covalent interactions.
  • the dextramer furthermore comprises fluorochrome coupled to the same dextran molecules through covalent or non-covalent interactions.
  • the MHC dextramers may all carry the same MHC I allele or carry different MHC I alleles.
  • Borrelia bacteria here include any strain of the species Borrelia Burgdorferi, Borrelia Garinii or Borrelia Afzelii.
  • Example Borrelia proteins include but is not limited to OspA, OspB, OspC, OspE, OspG, VIsE, FlaB, FIaA, BmpA, BmpB, BmpC, BmpD, HSP90, GRASP-1, CRASP-2, DbpA, DbpB, MaIQ, BapA, other outer surface Borrelia proteins, other intracellular proteins of Borrelia and plasmid encoded proteins from Borrelia bacteria.
  • the antigenic peptides are preferably 9′mers but can also be 8′mers, 10′ers or 11′mers.
  • the MHC dextramers are incubated for 1-60′ at room temperature in the dark, then gating reagents including labeled markers directed against CD3, CD8 and CD4 are added and incubation continued for 1-60′.
  • RBC is lysed by addition of lysis reagents e.g. EasyLyse then the sample is centrifuged, supernatant removed.
  • Cells are washed by resuspension in buffer followed by centrifugation. Supernatant is removed and cells resuspended in buffer.
  • fixative is added.
  • T cells specific for Borrelia antigens T cells specific for Borrelia antigens and their frequency can optionally be determined as follows.
  • the number of anti-CD8 and anti-CD3 stained cells and that of anti-CD8, MHC dextramer and anti-CD3 stained cells are determined.
  • the frequency of specific MHC dextramer binding cells, or antigen specific T-cells towards Borrelia specific peptide is calculated as “# of MHC dextramer positive, CD3+, CD8+cells/total # of CD3+, CD8 positive cells”.
  • A (sampling): Use of vacutainer or other container containing anti-coagulant to collect 4-10 ml human blood.
  • MHC dextramers are MHC-peptide complexes e.g. generated by in vitro refolding, coupled to dextran through covalent or non-covalent interactions.
  • the dextramer furthermore comprises fluorochrome coupled to the same dextran molecules through covalent or non-covalent interactions.
  • the MHC dextramers may all carry the same MHC I allele or carry different MHC I alleles.
  • Borrelia bacteria here include any strain of the species Borrelia Burgdorferi, Borrelia Garinii or Borrelia Afzelii.
  • Example Borrelia proteins include but is not limited to OspA, OspB, OspC, OspE, OspG, VIsE, FlaB, FIaA, BmpA, BmpB, BmpC, BmpD, HSP90, GRASP-1, CRASP-2, DbpA, DbpB, MaIQ, BapA, other outer surface Borrelia proteins, other intracellular proteins of Borrelia and plasmid encoded proteins from Borrelia bacteria.
  • the antigenic peptides are preferably 9′mers but can also be 8′mers, 10′ers or 11′mers.
  • MHC dextramers are incubated for 1-30′ at room temperature in the dark, then gating reagents including labeled markers directed against CD3, CD8 and CD4 are added and incubation continued for 1-60′.
  • RBC is lysed by addition of lysis reagents e.g. EasyLyse and buffer is added to dilute sample 2-50 times.
  • fixative is added.
  • T cells specific for Borrelia antigens T cells specific for Borrelia antigens and their frequency can optionally be determined as follows.
  • the number of anti-CD8 and anti-CD3 stained cells and that of anti-CD8, MHC dextramer and anti-CD3 stained cells are determined.
  • the frequency of specific MHC dextramer binding cells, or antigen specific T-cells towards Borrelia specific peptide is calculated as “# of MHC dextramer positive, CD3+, CD8+cells/total # of CD3+, CD8 positive cells”.
  • A (sampling): Use of vacutainer or other container containing anti-coagulant and cell separation medium to collect 4-10 ml human blood.
  • Example preparation Vacutainer is centrifuged thereby separating cells according to size and lymphocytes are transferred to a new container. A fraction or all purified lymphocytes are added one or more MHC dextramers labeled with fluorochrome and carrying antigenic peptides derived from proteins from Borrelia bacteria. The MHC dextramers may all carry the same MHC I allele or carry different MHC I alleles.
  • Borrelia bacteria here include any strain of the species Borrelia Burgdorferi, Borrelia Garinii or Borrelia Afzelii.
  • Example Borrelia proteins include but is not limited to OspA, OspB, OspC, OspE, OspG, VIsE, FlaB, FIaA, BmpA, BmpB, BmpC, BmpD, HSP90, GRASP-1, CRASP-2, DbpA, DbpB, MaIQ, BapA, other outer surface Borrelia proteins, other intracellular proteins of Borrelia and plasmid encoded proteins from Borrelia bacteria.
  • the antigenic peptides are preferably 9′mers but can also be 8′mers, 10′ers or 11′mers.
  • the sample and MHC dextramers are incubated for 1-30′ at room temperature in the dark, then gating reagents including labeled markers directed against CD3, CD8 and CD4 are added and incubation continued for 1-60′.
  • the sample is centrifuged and supernatant removed. Cells are washed by resuspension in buffer followed by centrifugation. Supernatant is removed and cells resuspended in buffer.
  • fixative is added.
  • T cells specific for Borrelia antigens T cells specific for Borrelia antigens and their frequency can optionally be determined as follows.
  • the number of anti-CD8 and anti-CD3 stained cells and that of anti-CD8, MHC dextramer and anti-CD3 stained cells are determined.
  • the frequency of specific MHC dextramer binding cells, or antigen specific T-cells towards Borrelia specific peptide is calculated as “# of MHC dextramer positive, CD3+, CD8+cells/total # of CD3+, CD8 positive cells”.
  • Process 11 Detection of Borrelia Specific T Cells in Sample of Human Blood Using a No-Lyse-No Wash Method.
  • A (sampling): Use of vacutainer or other container containing anti-coagulant to collect 4-10 ml human blood.
  • sample preparation The blood is transferred to tube containing cell separation medium, e.g. ficoll.
  • cell separation medium e.g. ficoll.
  • the tube is centrifuged thereby separating cells according to size and lymphocytes are transferred to a new container.
  • a fraction or all purified lymphocytes are added one or more MHC dextramers labeled with fluorochrome and carrying antigenic peptides derived from proteins from Borrelia bacteria.
  • Borrelia bacteria here include any strain of the species Borrelia Burgdorferi, Borrelia Garinii or Borrelia Afzelii.
  • Example Borrelia proteins include but is not limited to OspA, OspB, OspC, OspE, OspG, VIsE, FlaB, FIaA, BmpA, BmpB, BmpC, BmpD, HSP90, GRASP-1, CRASP-2, DbpA, DbpB, MaIQ, BapA, other outer surface Borrelia proteins, other intracellular proteins of Borrelia and plasmid encoded proteins from Borrelia bacteria.
  • the antigenic peptides are preferably 9′mers but can also be 8′mers, 10′ers or 11′mers.
  • sample and MHC dextramers are incubated for 1-30′ at room temperature in the dark, then gating reagents including labeled markers directed against CD3, CD8 and CD4 are added and incubation continued for 1-60′.
  • Buffer is added to dilute sample 2-50 times.
  • the sample is added fixative.
  • T cells specific for Borrelia antigens T cells specific for Borrelia antigens and their frequency can optionally be determined as follows.
  • the number of anti-CD8 and anti-CD3 stained cells and that of anti-CD8, MHC dextramer and anti-CD3 stained cells are determined.
  • the frequency of specific MHC dextramer binding cells, or antigen specific T-cells towards Borrelia specific peptide is calculated as “# of MHC dextramer positive, CD3+, CD8+cells/total # of CD3+, CD8 positive cells”.
  • Process 12 Detection of Borrelia Specific T Cells in Blood in a No-Lyse No-Wash Assay.
  • A (sampling): Use of vacutainer or other container containing anti-coagulant to collect 4-10 ml human blood.
  • sample preparation 10-500 ⁇ l blood is transferred to a tube and added one or more MHC dextramers labeled with fluorochrome and carrying antigenic peptides derived from proteins from Borrelia bacteria.
  • Borrelia bacteria here include any strain of the species Borrelia Burgdorferi, Borrelia Garinii or Borrelia Afzelii.
  • Example Borrelia proteins include but is not limited to OspA, OspB, OspC, OspE, OspG, VIsE, FlaB, FIaA, BmpA, BmpB, BmpC, BmpD, HSP90, GRASP-1, CRASP-2, DbpA, DbpB, MaIQ, BapA, other outer surface Borrelia proteins, other intracellular proteins of Borrelia and plasmid encoded proteins from Borrelia bacteria.
  • the antigenic peptides are preferably 9′mers but can also be 8′mers, 10′ers or 11′mers.
  • sample and MHC dextramers are incubated for 1-30′ at room temperature in the dark, then gating reagents including labeled markers directed against CD3, CD8, CD4, CD14 and CD45 are added and incubation continued for 1-60′.
  • markers are labeled with label different from MHC dextramer label and different from each other.
  • Anti-CD4 and anti-CD14 markers are “dump” gate markers and can be labeled with same fluorochrome.
  • Other dump gate markers like anti-CD19, anti-CD15 may also be included in analysis and are then labeled with same label as anti-CD4/anti-CD14 and added to the sample together with these markers.
  • Buffer is added to dilute sample 2-50 times.
  • an exact volume and known number of beads e.g. CytoCount beads can be added to the sample.
  • the sample is added fixative.
  • D data analysis: The obtained data are analyzed for the presences of cells stained with MHC dextramer, anti-CD3, anti-CD8 as follows. Cells stained positive with anti-CD4 and anti-CD14 are deselected in a “dump” gate and remaining cells stained positive with anti-CD45 are selected for further analysis. In this population the presences of cells stained with MHC dextramer, anti-CD3 and anti-CD8 markers are determined.
  • the amount of acquired beads is also determined.
  • E (interpretation of data): The presence of cells stained with MHC dextramer, anti-CD3 and anti-CD8 are T cells specific for Borrelia antigens and their frequency can optionally be determined as follows. The number of anti-CD8 and anti-CD3 stained cells and that of anti-CD8, MHC dextramer and anti-CD3 stained cells are determined. The frequency of specific MHC dextramer binding cells, or antigen specific T-cells towards Borrelia specific peptide is calculated as “# of MHC dextramer positive, CD3+, CD8+cells/total # of CD3+, CD8 positive cells”.
  • Concentration of Borrelia specific T cells Dilution factor of blood ⁇ (# counted specific T cells ⁇ concentration of bead)/# counted beads.
  • Process 13 Detection of Borrelia Specific T Cells in Blood in a No-Lyse No-Wash Assay Using a Tube Containing Solidified Reagents.
  • A (sampling): Use of vacutainer or other tube containing anti-coagulant to collect 4-10 ml human blood.
  • sample preparation 10-500 ⁇ l blood is transferred to a tube containing a solid or semisolid matrix featuring one or more MHC dextramers labeled with fluorochrome and carrying antigenic peptides derived from proteins from Borrelia bacteria.
  • Borrelia bacteria here include any strain of the species Borrelia Burgdorferi, Borrelia Garinii or Borrelia Afzelii.
  • Example Borrelia proteins include but is not limited to OspA, OspB, OspC, OspE, OspG, VIsE, FlaB, FIaA, BmpA, BmpB, BmpC, BmpD, HSP90, GRASP-1, CRASP-2, DbpA, DbpB, MaIQ, BapA, other outer surface Borrelia proteins, other intracellular proteins of Borrelia and plasmid encoded proteins from Borrelia bacteria.
  • the antigenic peptides are preferably 9′mers but can also be 8′mers, 10′ers or 11′mers.
  • the matrix also features gating reagents including labeled markers directed against CD3, CD8, CD4, CD14 and CD45.
  • markers are labeled with label different from MHC dextramer label and different from each other.
  • Anti-CD4 and anti-CD14 markers are “dump” gate markers and can be labeled with same fluorochrome.
  • Other dump-gate markers like anti-CD19, anti-CD15 or others may also be included in analysis and are then labeled with same label as anti-CD4/anti-CD14 markers and are also contained in matrix material.
  • the matrix material is such that MHC dextramers and gating reagents are released upon addition of blood.
  • the matrix material is dissolved thereby releasing reagents into added sample.
  • the sample is incubated for 1-60′ at room temperature in the dark.
  • additional gating reagents can be added. Buffer is added to dilute sample 2-50 times.
  • an exact volume and known number of beads e.g. CytoCount beads can be added to the sample.
  • the sample is added fixative.
  • D data analysis: The obtained data are analyzed for the presences of cells stained with MHC dextramer, anti-CD3, anti-CD8 as follows. Cells stained positive with anti-CD4 and anti-CD14 are deselected in a “dump” gate and remaining cells stained positive with anti-CD45 are selected for further analysis. In this population the presences of cells stained with MHC dextramer, anti-CD3 and anti-CD8 markers are determined.
  • the amount of acquired beads is also determined.
  • E (interpretation of data): The presence of cells stained with MHC dextramer, anti-CD3 and anti-CD8 are T cells specific for Borrelia antigens and their frequency can optionally be determined as follows. The number of anti-CD8 and anti-CD3 stained cells and that of anti-CD8, MHC dextramer and anti-CD3 stained cells are determined. The frequency of specific MHC dextramer binding cells, or antigen specific T-cells towards Borrelia specific peptide is calculated as “# of MHC dextramer positive, CD3+, CD8+cells/total # of CD3+, CD8 positive cells”.
  • Concentration of Borrelia specific T cells Dilution factor of blood ⁇ (# counted specific T cells ⁇ concentration of bead)/# counted beads.
  • Process 14 Determination of Absolute Count of CD3+, CD4+and CD3+, CD8+T Cell Subsets in Human Blood.
  • A (sampling): Use of vacutainer or other tube containing anti-coagulant to collect 4-10 ml human blood.
  • sample preparation 10-500 ⁇ l blood is transferred to a tube together with anti-CD3, anti-CD4 and anti-CD8 markers labeled with different fluorochromes.
  • the sample is incubated for 1-60′ at room temperature in the dark and lysis reagent is added, e.g. EasyLyse or iTAG MHC Tetramer Lyse reagent. Following incubation buffer is added to dilute sample 2-50 times.
  • the sample is added fixative.
  • D data analysis: The obtained data are analyzed for the presences of cells stained with anti-CD3 and anti-CD8 markers and cells stained with anti-CD3 and anti-CD4 markers.
  • the presence and amount of beads acquired is also analyzed.
  • E (interpretation of data): The number of anti-CD8+, anti-CD3 double positive cells and the number of anti-CD4+, anti-CD3+double positive cells acquired are determined.
  • the absolute concentration of CD3+, CD8+positive cells can be calculated as follows:
  • Concentration of CD 3+, CD 8+positive cells (# counted CD 3+, CD 8+positive cells ⁇ concentration of bead)/# counted beads.
  • CD3+ CD4+positive cells
  • Concentration of CD 3+, CD 4+positive cells (# counted CD 3+, CD 4+positive cells ⁇ concentration of bead)/# counted beads.
  • Process 15 Determination of Absolute Count of Borrelia Specific T Cells in Human Blood Using a Two-Panel Technique.
  • A (sampling): Use of vacutainer or other tube containing anti-coagulant to collect 4-10 ml human blood.
  • sample preparation 10-500 ⁇ l blood is transferred to a two tubes.
  • the first tube is added anti-CD3, anti-CD4 and anti-CD8 markers labeled with different fluorochromes either before or after addition of blood.
  • the second tube are added anti-CD3, anti-CD4 and anti-CD8 markers labeled with different fluorochromes and also contains one or more MHC dextramers labeled with fluorochrome and carrying antigenic peptides derived from proteins from Borrelia bacteria.
  • the label of the individual MHC dextramers are either the same or different from each other. However all MHC dextramers are labeled with label(s) different for the labels used for the other marker molecules.
  • Borrelia bacteria here include any strain of the species Borrelia Burgdorferi, Borrelia Garinii or Borrelia Afzelii.
  • Example Borrelia proteins include but is not limited to OspA, OspB, OspC, OspE, OspG, VIsE, FlaB, FIaA, BmpA, BmpB, BmpC, BmpD, HSP90, CRASP-1, CRASP-2, DbpA, DbpB, MaIQ, BapA, other outer surface Borrelia proteins, other intracellular proteins of Borrelia and plasmid encoded proteins from Borrelia bacteria.
  • the antigenic peptides are preferably 9′mers but can also be 8′mers, 10′ers or 11′mers.
  • Both tubes are incubated for 1-60′ at room temperature in the dark and lysis reagent is added, e.g. EasyLyse or iTAG MHC Tetramer Lyse reagent.
  • lysis reagent e.g. EasyLyse or iTAG MHC Tetramer Lyse reagent.
  • the lysis reagent may optionally contain fixative.
  • Tube two is centrifuged, supernatant removed and buffer added to wash cells.
  • D data analysis: The obtained data for tube 1 are analyzed for the presences of cells stained with anti-CD3 and anti-CD8 markers and cells stained with anti-CD3 and anti-CD4 markers. The presence and amount of beads acquired is also analyzed.
  • the obtained data for tube 2 are analyzed for the presence of cells stained with anti-CD3 and anti-CD8 markers, cells stained with anti-CD3 and anti-CD4 markers and also cells triple stained with MHC dextramers, anti-CD3 and anti-CD8 markers.
  • E (interpretation of data): The number of anti-CD8+, anti-CD3 double positive cells and the number of anti-CD4+, anti-CD3+double positive cells acquired in tube one and tube 2 are determined. The number of acquired beads in tube one is also determined.
  • the absolute concentration of CD3+, CD8+positive cells can be calculated as follows:
  • Concentration of CD 3+, CD 8+positive cells (# counted CD 3+, CD 8+positive cells ⁇ concentration of bead)/# counted beads.
  • the frequency of specific MHC dextramer binding cells, or antigen specific T-cells towards Borrelia specific peptide is calculated as: (# of MHC dextramer positive, CD3+ and CD8+cells)/(total # of CD3+, CD8+positive cells).
  • Absolute concentration of Borrelia specific T cells can then be calculated as follows:
  • Process 16 Determination of Presences of Borrelia Specific T Cells in Human Blood Including a Negative Control Reagent.
  • A (sampling): Use of vacutainer or other container containing anti-coagulant to collect 4-10 ml human blood.
  • MHC dextramers are MHC-peptide complexes e.g. generated by in vitro refolding, coupled to dextran through covalent or non-covalent interactions.
  • the dextramer furthermore comprises fluorochrome coupled to the same dextran molecules through covalent or non-covalent interactions.
  • the MHC dextramers may all carry the same MHC I allele or carry different MHC I alleles.
  • Borrelia bacteria here include any strain of the species Borrelia Burgdorferi, Borrelia Garinii or Borrelia Afzelii.
  • Example Borrelia proteins include but is not limited to OspA, OspB, OspC, OspE, OspG, VIsE, FlaB, FIaA, BmpA, BmpB, BmpC, BmpD, HSP90, GRASP-1, CRASP-2, DbpA, DbpB, MaIQ, BapA, other outer surface Borrelia proteins, other intracellular proteins of Borrelia and plasmid encoded proteins from Borrelia bacteria.
  • Negative control peptides are peptides binding the MHC allele of choice, but that does not support binding of the resultant MHC-peptide complex to the desired TCR.
  • Example negative control peptides include but are not limited to modified peptides, naturally occurring peptide different from the peptide used for analysis and nonsense peptides (peptides with an amino acid sequence different from the linear sequence of any peptide derived from any known protein).
  • the antigenic peptides and/or negative control peptides are preferably 9′mers but can also be 8′mers, 10′ers or 11′mers.
  • Cells are washed by resuspension in buffer followed by centrifugation. Supernatant is removed and cells resuspended in buffer.
  • fixative is added.
  • T cells specific for Borrelia antigens are T cells specific for Borrelia antigens and their frequency can optionally be determined as follows.
  • the number of anti-CD8 and anti-CD3 stained cells and that of anti-CD8, MHC dextramer and anti-CD3 stained cells are determined.
  • the frequency of specific MHC dextramer binding cells, or antigen specific T-cells towards Borrelia specific peptide is calculated as “# of MHC dextramer positive, CD3+and CD8+triple positive cells/total # of CD3+,CD8+ double positive cells”.
  • Negative control reagents comprising MHC dextramers carrying negative control peptides can be included in all processes involving staining with MHC dextramers.
  • A (sampling): Use of vacutainer or other container containing anti-coagulant to collect 4-10 ml human blood.
  • MHC dextramers are MHC-peptide complexes e.g. generated by in vitro refolding, coupled to dextran through covalent or non-covalent interactions.
  • the dextramer furthermore comprises fluorochrome coupled to the same dextran molecules through covalent or non-covalent interactions.
  • the MHC dextramers may all carry the same MHC II allele or carry different MHC II alleles.
  • Borrelia bacteria here include any strain of the species Borrelia Burgdorferi, Borrelia Garinii or Borrelia Afzelii.
  • Example Borrelia proteins include but is not limited to OspA, OspB, OspC, OspE, OspG, VIsE, FlaB, FIaA, BmpA, BmpB, BmpC, BmpD, HSP90, GRASP-1, CRASP-2, DbpA, DbpB, MaIQ, BapA, other outer surface Borrelia proteins, other intracellular proteins of Borrelia and plasmid encoded proteins from Borrelia bacteria.
  • the antigenic peptides are preferably 12-18′mers but can also be 9′mers, 10′ers, 11′mers, 19′mers, 20′mers or even longer peptide fragments.
  • the MHC dextramers are incubated for 1-60′ at room temperature in the dark, then gating reagents including labeled markers directed against CD3, CD8 and CD4 are added and incubation continued for 1-60′.
  • RBC is lysed by addition of lysis reagents e.g. EasyLyse then the sample is centrifuged, supernatant removed.
  • Cells are washed by resuspension in buffer followed by centrifugation. Supernatant is removed and cells resuspended in buffer.
  • fixative is added.
  • D data analysis: The obtained data are analyzed for the presences of cells stained with MHC dextramer, anti-CD3, anti-CD4 and not stained with anti-CD8.
  • T cells specific for Borrelia antigens are T cells specific for Borrelia antigens and their frequency can optionally be determined as follows.
  • the number of anti-CD4 and anti-CD3 stained cells and that of anti-CD4, MHC dextramer and anti-CD3 stained cells are determined.
  • the frequency of specific MHC dextramer binding cells, or antigen specific T-cells towards Borrelia specific peptide is calculated as “# of MHC dextramer positive, CD3+, CD4+cells/total # of CD3+, CD4 positive cells”.
  • A (sampling): Use of vacutainer or other container containing anti-coagulant to collect 4-10 ml human blood.
  • MHC dextramers are MHC-peptide complexes e.g. generated by in vitro refolding, coupled to dextran through covalent or non-covalent interactions.
  • the dextramer furthermore comprises fluorochrome coupled to the same dextran molecules through covalent or non-covalent interactions.
  • the MHC dextramers may all carry the same MHC II allele or carry different MHC II alleles.
  • Borrelia bacteria here include any strain of the species Borrelia Burgdorferi, Borrelia Garinii or Borrelia Afzelii.
  • Example Borrelia proteins include but is not limited to OspA, OspB, OspC, OspE, OspG, VIsE, FlaB, FIaA, BmpA, BmpB, BmpC, BmpD, HSP90, GRASP-1, CRASP-2, DbpA, DbpB, MaIQ, BapA, other outer surface Borrelia proteins, other intracellular proteins of Borrelia and plasmid encoded proteins from Borrelia bacteria.
  • the antigenic peptides are preferably 12-18′mers but can also be 9′mers, 10′ers, 11′mers, 19′mers, 20′mers or even longer peptide fragments.
  • MHC dextramers are incubated for 1-30′ at room temperature in the dark, then gating reagents including labeled markers directed against CD3, CD8 and CD4 are added and incubation continued for 1-60′.
  • RBC is lysed by addition of lysis reagents e.g. EasyLyse and buffer is added to dilute sample 2-50 times.
  • fixative is added.
  • D data analysis: The obtained data are analyzed for the presences of cells stained with MHC dextramer, anti-CD3, anti-CD4 and not stained with anti-CD8.
  • T cells specific for Borrelia antigens are T cells specific for Borrelia antigens and their frequency can optionally be determined as follows.
  • the number of anti-CD4 and anti-CD3 stained cells and that of anti-CD4, MHC dextramer and anti-CD3 stained cells are determined.
  • the frequency of specific MHC dextramer binding cells, or antigen specific T-cells towards Borrelia specific peptide is calculated as “# of MHC dextramer positive, CD3+, CD4+cells/total # of CD3+, CD4 positive cells”.
  • A (sampling): Use of vacutainer or other container containing anti-coagulant and cell separation medium to collect 4-10 ml human blood.
  • Example preparation Vacutainer is centrifuged thereby separating cells according to size and lymphocytes are transferred to a new container. A fraction or all purified lymphocytes are added one or more MHC dextramers labeled with fluorochrome and carrying antigenic peptides derived from proteins from Borrelia bacteria. The MHC dextramers may all carry the same MHC II allele or carry different MHC II alleles.
  • Borrelia bacteria here include any strain of the species Borrelia Burgdorferi, Borrelia Garinii or Borrelia Afzelii.
  • Example Borrelia proteins include but is not limited to OspA, OspB, OspC, OspE, OspG, VIsE, FlaB, FIaA, BmpA, BmpB, BmpC, BmpD, HSP90, GRASP-1, CRASP-2, DbpA, DbpB, MaIQ, BapA, other outer surface Borrelia proteins, other intracellular proteins of Borrelia and plasmid encoded proteins from Borrelia bacteria.
  • the antigenic peptides are preferably 12-18′mers but can also be 9′mers, 10′ers, 11′mers, 19′mers, 20′mers or even longer peptide fragments.
  • the sample and MHC dextramers are incubated for 1-30′ at room temperature in the dark, then gating reagents including labeled markers directed against CD3, CD8 and CD4 are added and incubation continued for 1-60′.
  • the sample is centrifuged and supernatant removed. Cells are washed by resuspension in buffer followed by centrifugation. Supernatant is removed and cells resuspended in buffer.
  • fixative is added.
  • D data analysis: The obtained data are analyzed for the presences of cells stained with MHC dextramer, anti-CD3, anti-CD4 and not stained with anti-CD8.
  • T cells specific for Borrelia antigens are T cells specific for Borrelia antigens and their frequency can optionally be determined as follows.
  • the number of anti-CD4 and anti-CD3 stained cells and that of anti-CD4, MHC dextramer and anti-CD3 stained cells are determined.
  • the frequency of specific MHC dextramer binding cells, or antigen specific T-cells towards Borrelia specific peptide is calculated as “# of MHC dextramer positive, CD3+, CD4+cells/total # of CD3+, CD4 positive cells”.
  • A (sampling): Use of vacutainer or other container containing anti-coagulant to collect 4-10 ml human blood.
  • sample preparation The blood is transferred to tube containing cell separation medium, e.g. ficoll.
  • cell separation medium e.g. ficoll.
  • the tube is centrifuged thereby separating cells according to size and lymphocytes are transferred to a new container.
  • a fraction or all purified lymphocytes are added one or more MHC dextramers labeled with fluorochrome and carrying antigenic peptides derived from proteins from Borrelia bacteria.
  • Borrelia bacteria here include any strain of the species Borrelia Burgdorferi, Borrelia Garinii or Borrelia Afzelii.
  • Example Borrelia proteins include but is not limited to OspA, OspB, OspC, OspE, OspG, VIsE, FlaB, FIaA, BmpA, BmpB, BmpC, BmpD, HSP90, GRASP-1, CRASP-2, DbpA, DbpB, MaIQ, BapA, other outer surface Borrelia proteins, other intracellular proteins of Borrelia and plasmid encoded proteins from Borrelia bacteria.
  • the antigenic peptides are preferably 12-18′mers but can also be 9′mers, 10′ers, 11′mers, 19′mers, 20′mers or even longer peptide fragments.
  • sample and MHC dextramers are incubated for 1-30′ at room temperature in the dark, then gating reagents including labeled markers directed against CD3, CD8 and
  • CD4 are added and incubation continued for 1-60′.
  • Buffer is added to dilute sample 2-50 times.
  • the sample is added fixative.
  • D data analysis: The obtained data are analyzed for the presences of cells stained with MHC dextramer, anti-CD3, anti-CD4 and not stained with anti-CD8.
  • T cells specific for Borrelia antigens are T cells specific for Borrelia antigens and their frequency can optionally be determined as follows.
  • the number of anti-CD4 and anti-CD3 stained cells and that of anti-CD4, MHC dextramer and anti-CD3 stained cells are determined.
  • the frequency of specific MHC dextramer binding cells, or antigen specific T-cells towards Borrelia specific peptide is calculated as “# of MHC dextramer positive, CD3+, CD4+cells/total # of CD3+, CD4 positive cells”.
  • Process 21 Detection of Borrelia Specific T Cells in Blood in a No-Lyse No-Wash Assay.
  • A (sampling): Use of vacutainer or other container containing anti-coagulant to collect 4-10 ml human blood.
  • sample preparation 10-500 ⁇ l blood is transferred to a tube and added one or more MHC dextramers labeled with fluorochrome and carrying antigenic peptides derived from proteins from Borrelia bacteria.
  • Borrelia bacteria here include any strain of the species Borrelia Burgdorferi, Borrelia Garinii or Borrelia Afzelii.
  • Example Borrelia proteins include but is not limited to OspA, OspB, OspC, OspE, OspG, VIsE, FlaB, FIaA, BmpA, BmpB, BmpC, BmpD, HSP90, GRASP-1, CRASP-2, DbpA, DbpB, MaIQ, BapA, other outer surface Borrelia proteins, other intracellular proteins of Borrelia and plasmid encoded proteins from Borrelia bacteria.
  • the antigenic peptides are preferably 12-18′mers but can also be 9′mers, 10′ers, 11′mers, 19′mers, 20′mers or even longer peptide fragments.
  • sample and MHC dextramers are incubated for 1-30′ at room temperature in the dark, then gating reagents including labeled markers directed against CD3, CD8, CD4, CD14 and CD45 are added and incubation continued for 1-60′.
  • markers are labeled with label different from MHC dextramer label and different from each other.
  • Anti-CD8 and anti-CD14 markers are “dump” gate markers and can be labeled with same fluorochrome.
  • Other dump gate markers like anti-CD19, anti-CD15 may also be included in analysis and are then labeled with same label as anti-CD8/anti-CD14 and added to the sample together with these markers.
  • Buffer is added to dilute sample 2-50 times.
  • an exact volume and known number of beads e.g. CytoCount beads can be added to the sample.
  • the sample is added fixative.
  • D data analysis: The obtained data are analyzed for the presences of cells stained with MHC dextramer, anti-CD3, anti-CD4 as follows. Cells stained positive with anti-CD8 and anti-CD14 are deselected in a “dump” gate and remaining cells stained positive with anti-CD45 are selected for further analysis. In this population the presences of cells stained with MHC dextramer, anti-CD3 and anti-CD4 markers are determined.
  • the amount of acquired beads is also determined.
  • E (interpretation of data): The presence of cells stained with MHC dextramer, anti-CD3 and anti-CD4 are T cells specific for Borrelia antigens and their frequency can optionally be determined as follows. The number of anti-CD4 and anti-CD3 stained cells and that of anti-CD4, MHC dextramer and anti-CD3 stained cells are determined.
  • the frequency of specific MHC dextramer binding cells, or antigen specific T-cells towards Borrelia specific peptide is calculated as “# of MHC dextramer positive, CD3+, CD4+cells/total # of CD3+, CD4 positive cells”.
  • Concentration of Borrelia specific T cells Dilution factor of blood ⁇ (# counted specific T cells ⁇ concentration of bead)/# counted beads.
  • Process 22 Determination of Absolute Count of Borrelia Specific T Cells in Human Blood Using a Two-Panel Technique.
  • A (sampling): Use of vacutainer or other tube containing anti-coagulant to collect 4-10 ml human blood.
  • sample preparation 10-500 ⁇ l blood is transferred to a two tubes.
  • the first tube is added anti-CD3, anti-CD4 and anti-CD8 markers labeled with different fluorochromes either before or after addition of blood.
  • the second tube are added anti-CD3, anti-CD4 and anti-CD8 markers labeled with different fluorochromes and also contains one or more MHC dextramers labeled with fluorochrome and carrying antigenic peptides derived from proteins from Borrelia bacteria.
  • the label of the individual MHC dextramers are either the same or different from each other. However all MHC dextramers are labeled with label(s) different for the labels used for the other marker molecules.
  • Borrelia bacteria here include any strain of the species Borrelia Burgdorferi, Borrelia Garinii or Borrelia Afzelii.
  • Example Borrelia proteins include but is not limited to OspA, OspB, OspC, OspE, OspG, VIsE, FlaB, FIaA, BmpA, BmpB, BmpC, BmpD, HSP90, CRASP-1, CRASP-2, DbpA, DbpB, MaIQ, BapA, other outer surface Borrelia proteins, other intracellular proteins of Borrelia and plasmid encoded proteins from Borrelia bacteria.
  • the antigenic peptides are preferably 12-18′mers but can also be 9′mers, 10′ers, 11′mers, 19′mers, 20′mers or even longer peptide fragments.
  • Both tubes are incubated for 1-60′ at room temperature in the dark and lysis reagent is added, e.g. EasyLyse or iTAG MHC Tetramer Lyse reagent.
  • lysis reagent e.g. EasyLyse or iTAG MHC Tetramer Lyse reagent.
  • the lysis reagent may optionally contain fixative.
  • Tube two is centrifuged, supernatant removed and buffer added to wash cells.
  • D data analysis: The obtained data for tube 1 are analyzed for the presences of cells stained with anti-CD3 and anti-CD8 markers and cells stained with anti-CD3 and anti-CD4 markers. The presence and amount of beads acquired is also analyzed.
  • the obtained data for tube 2 are analyzed for the presence of cells stained with anti-CD3 and anti-CD8 markers, cells stained with anti-CD3 and anti-CD4 markers and also cells triple stained with MHC dextramers, anti-CD3 and anti-CD4 markers.
  • E The number of anti-CD8+, anti-CD3 double positive cells and the number of anti-CD4+, anti-CD3+ double positive cells acquired in tube one and tube 2 are determined. The number of acquired beads in tube one is also determined. The absolute concentration of CD3+, CD8+ positive cells can be calculated as follows:
  • Concentration of CD 3+, CD 8+positive cells (# counted CD 3+, CD 8+positive cells ⁇ concentration of bead)/# counted beads.
  • the frequency of specific MHC dextramer binding cells, or antigen specific T-cells towards Borrelia specific peptide is calculated as: (# of MHC dextramer positive, CD3+ and CD4+ cells)/(total # of CD3+, CD4+ positive cells).
  • Absolute concentration of Borrelia specific T cells can then be calculated as follows:
  • MHC tetramers carrying Borrelia specific peptides.
  • MHC tetramers are Streptavidin or Avidin associated with 4 biotinylated MHC-peptide molecules and labeled with flourochrome e.g. APC or PE.
  • Preferred processes involving use of MHC tetramers carrying Borrelia specific peptides for detection of Borrelia specific T cells include processes similar to process 7-13, 15-22 but where MHC tetramers are used in stead of MHC dextramers.
  • MHC multimers carrying Borrelia specific peptides.
  • MHC multimers are complexes comprising of more than one MHC-peptide complexes, held together by covalent or non-covalent interactions between a multimerization domain and one or more MHC-peptide complexes and where the multimerizations domain is a molecule, a complex of molecules, a cell or a solid support.
  • Preferred processes involving use of MHC multimers carrying Borrelia specific peptides for detection of Borrelia specific T cells include processes similar to process 7-13, 15-22 but where MHC multimers are used instead of MHC dextramers.
  • A (sampling): Use of vacutainer or other container containing anti-coagulant to collect 4-10 ml human blood.
  • sample preparation 100 ⁇ l of the full blood is transferred to a tube containing one or more MHC dextramers labeled with APC and carrying antigenic peptides derived from proteins from human Cytomegalovirus (CMV).
  • CMV Cytomegalovirus
  • the MHC dextramers may all carry the same MHC I allele or carry different MHC I alleles.
  • Example CMV proteins include but is not limited to Matrix phosphoprotein 65 (pp65), DNA polymerase, immediate early protein 1 (IE1), immediate early protein 3 (IE3), any intracellular protein, any surface protein or any other protein from which peptide epitopes are presented by MHC I molecules.
  • pp65 Matrix phosphoprotein 65
  • DNA polymerase DNA polymerase
  • IE1 immediate early protein 1
  • IE3 immediate early protein 3
  • intracellular protein any surface protein or any other protein from which peptide epitopes are presented by MHC I molecules.
  • antigenic peptides are preferably 9′mers but can also be 8′mers, 10′ers or 11′mers.
  • Example of antigenic peptides include but is not limited to NLVPMVATV binding the MHC I allele HLA-A*0201, VTEHDTLLY binding HLA-A*0101, QYDPVAALF binding HLA-A*2402, TPRVTGGGAM binding B*0702, ELRRKMMYM binding B*0801, IPSINVHHY binding
  • the MHC dextramers are incubated for 10′ at room temperature in the dark, then gating reagents including anti-CD3/PE, anti-CD8/PB and anti-CD4/FITC are added and incubation continued for 20′.
  • RBC is lysed by addition of lysis reagents e.g. EasyLyse then the sample is centrifuged, supernatant removed. Cells are washed by resuspension in buffer followed by centrifugation. Supernatant is removed and cells resuspended in buffer.
  • fixative is added.
  • E (interpretation of data): The presence of cells stained with MHC dextramer, anti-CD3, anti-CD8 and not stained with anti-CD4 are T cells specific for CMV antigens and their frequency can optionally be determined as follows. The number of anti-CD8 and anti-CD3 stained cells and that of anti-CD8, MHC dextramer and anti-CD3 stained cells are determined. The frequency of specific MHC dextramer binding cells, or antigen specific T-cells towards CMV specific peptide is calculated as “# of MHC dextramer positive, CD3+, CD8+ cells/total # of CD3+,CD8 positive cells”.
  • A (sampling): Use of vacutainer or other container containing anti-coagulant to collect 4-10 ml human blood.
  • sample preparation 10-500 ⁇ l of the full blood is transferred to a tube containing one or more MHC dextramers labeled with fluorochrome and carrying antigenic peptides derived from proteins from CMV.
  • the MHC dextramers may all carry the same MHC I allele or carry different MHC I alleles.
  • Example CMV proteins include but is not limited to Matrix phosphoprotein 65 (pp65), DNA polymerase, immediate early protein 1 (IE1), immediate early protein 3 (IE3), any intracellular protein, any surface protein or any other protein from which peptide epitopes are presented by MHC I molecules.
  • pp65 Matrix phosphoprotein 65
  • DNA polymerase DNA polymerase
  • IE1 immediate early protein 1
  • IE3 immediate early protein 3
  • intracellular protein any surface protein or any other protein from which peptide epitopes are presented by MHC I molecules.
  • antigenic peptides are preferably 9′mers but can also be 8′mers, 10′ers or 11′mers.
  • Example of antigenic peptides include but is not limited to NLVPMVATV binding the MHC I allele HLA-A*0201, VTEHDTLLY binding HLA-A*0101, QYDPVAALF binding HLA-A*2402, TPRVTGGGAM binding B*0702, ELRRKMMYM binding B*0801, IPSINVHHY binding B*3501, AYAQKIFKI binding HLA-A*2402, KLGGALQAK binding HLA-A*0301, VYALPLKML binding HLA-A*2402 and RPHERNGFTVL binding HLA-B*0702.
  • the MHC dextramers are incubated for 1-60′ at room temperature in the dark, then gating reagents including labeled markers directed against CD3, CD8 and CD4 are added and incubation continued for 1-60′.
  • RBC is lysed by addition of lysis reagents e.g. EasyLyse then the sample is centrifuged, supernatant removed.
  • Cells are washed by resuspension in buffer followed by centrifugation. Supernatant is removed and cells resuspended in buffer.

Abstract

The present invention relates to the fields of analysis, diagnostics, prognostics, standardization, characterization and enumeration of entities such as biological cells, as well as therapeutical applications. Accordingly, the present invention in preferred aspects is directed to methods for the detection and/or analysis and/or isolation and optionally further manipulation of entities, such as cells, particles, and supra-molecular structures.

Description

  • This application claims priority under 35 U.S.C. §120 as a continuation of PCT Application No. PCT/DK2008/050168 filed Jul. 3, 2008, which claims priority to the following Danish Patent applications Nos.—PA 2007 00972, filed Jul. 3, 2007, PA 2007 00973, filed Jul. 3, 2007, PA 2007 00974, filed Jul. 3, 2007, and PA 2007 00975, filed Jul. 3, 2007, and also claims priority to the following U.S. Provisional Patent Applications Nos.—U.S. 60/929,581, filed Jul. 3, 2007, U.S. 60/929,582, filed Jul. 3, 2007, U.S. 60/929,583, filed Jul. 3, 2007, and U.S. 60/929,586, Jul. 3, 2007, the contents of each of which are hereby incorporated by reference.
  • All patent and non-patent references cited in this application are hereby incorporated by reference in their entirety. All patent and non-patent references cited in U.S. 60/929,583, U.S. 60/929,586, U.S. 60/929,582, U.S. 60/929,581, PA 2007 00974, PA 2007 00972 and PA 2007 00975 are hereby incorporated by reference in their entirety. U.S. 60/929,583, U.S. 60/929,586, U.S. 60/929,582, U.S. 60/929,581, PA 2007 00974, PA 2007 00972 and PA 2007 00975 are hereby also incorporated herein by reference in its entirety.
    • FIELD OF INVENTION
  • The present invention relates to methods for assaying an entity present in a sample. The method comprises a number of individual steps, such as the steps of obtaining a sample, preparing the sample for assaying, and assaying the sample. The method can comprise further optional processing steps associated with either sample processing or processing of data obtained as a result of having assayed the sample.
  • The sample can initially be assayed e.g. for presence of one or more entities. The step of assaying the sample can further comprise the step of analyzing one or more characteristics of the one or more entities. On the basis of the determination of the presence of the one or more entities in the sample, or the result of an analysis of the one or more entities, the method can comprise one or more further processing steps, such as data processing steps and/or sample processing steps associated with partitioning and/or isolation of the one or more entities in the sample which was subjected to the assaying procedure.
    • BACKGROUND OF INVENTION
  • A number of methods exist for the analysis and sorting of entities such as cells, supramolecular compartments and particles. Analytical principles include binding to a solid support e.g affinity columns, flow cytometry and stationary cytometry such as immunohistochemistry. However, often the process of sampling, sample preparation, analysis or sorting, and data acquisition is not adequately optimized with the specific goal in mind. Sometimes not even the analysis itself is optimized adequately. As an example, flow cytometry analyses often include reagents that have not been properly design to achieve the best possible result: The individual reagents and their binding affinity or target abundance is not seriously considered and corrected for, and likewise, the fluorochromes chosen are not well enough separated as regards wavelength of maximum emission, and therefore, the spectra of the fluorochormes significantly overlap, blurring the data further.
    • SUMMARY OF THE INVENTION
  • The present invention relates to the fields of analysis, diagnostics, prognostics, standardization, characterization and enumeration of entities such as biological cells, as well as therapeutical applications.
  • Accordingly, the present invention in preferred aspects is directed to methods for the detection and/or analysis and/or isolation and optionally further manipulation of entities, such as cells, particles, and supra-molecular structures.
  • In one preferred aspect of the present invention there is provided a method for detecting and/or analyzing and/or partitioning one or more entities potentially present in a sample, said method comprising the steps of
      • A) providing a sample by extracting the sample from a sample source,
      • B) preparing the sample for assaying by contacting said sample with one or more of i) a marker molecule specific for the one or more entities; ii) a labelling molecule specific for the one or more entities; and iii) a detection molecule comprising a marker molecule and a labelling molecule specific for the one or more entities, and
      • C) assaying the sample at least for the presence of the one or more entities.
  • The method can comprise the further steps of D) data processing and/or sample processing, such as sample analysis and/or partitioning, as well as E) data interpretation and/or further sample manipulations, such as cultivation and/or expansion of partitioned and/or isolated cells.
  • Particularly interesting marker molecules in accordance with the present invention are MHC molecules and MHC-multimers. MHC-multimers are complexes comprising multiple MHC molecules, such as MHC-peptide complexes comprising said MHC molecules.
    • BRIEF DESCRIPTION OF THE FIGURES
  • FIG. 1: Example cell-antigens, for which marker molecules is antibodies or other molecules able to specifically bind the cell-antigen. The table primarily shows cell-antigens able to differentiate blood cell populations. The cell-antigens are grouped for the cell type by which they are expressed. Also is shown which preferable antigens markers can be directed against and then used as contra or “dump” markers or as pro selective markers in gating strategies when analyzing samples with MHC multimers. The two columns at the right show cell-antigens important for identification of cell-“status”, e.g. activation, differentiation state.
  • FIG. 2: Gating strategy for identification and enumeration of antigen specific cytotoxic T cells
  • 2A; Negative control using a HIV specific peptide loaded into MHC dextramers
  • 2B: MHC dextramers loaded with a CMV specific peptide
  • The method used in this example is a “No lyse” method, meaning that the sample red blood cells (RBC) is not lysed during cell preparation resulting in poorly defined scatter picture. Therefore the “RAW cell gate” is poorly defined in scatter parameters. The scatter gated cells are further “purified” by a anti-CD3, anti-CD8 gate, transferring only the CD3 and CD8 positive cells, to the histogram/dot plot where the MHCCMV positive cells can be read off. In this example the labeling molecules used for the markers, anti-CD45, anti-CD3, anti-CD8 antibodies and for the MHC dextramers are, Cascade Yellow (CY), Pasific Blue (PB), Alexa700 and PE, respectively.
  • Counting beads was added to the sample and can be selected in different ways when analyzing the samples, e.g. using that the bead has low FSC compared to cells, or that the beads have high fluorescence signals detectable with Cascade yellow and FITC detectors. In this example the beads was selected for analysis by using the FITC detector.
  • The amount of counting beads added to the sample was known and thereby the concentration of beads in the sample. Gate R8 gives the bead count in the acquired part of the sample and from this number the volume of sample acquired may be calculated.
  • The gates are defined as follows: R8 define the beads, and is subtracted out in all gates used except in Gate1. Gate 1(R1) define the Raw cell gate in FSC/SSC plot, Gate 2, define the cells that also have High Alexa 700 and PB signals, i.e are CD8 and CD3 positive, Gate 3; restricts the CD8 positive cells between which the CMV specific cytotoxic T cells will be found.
  • FIG. 3: Scatter defined blood cell populations
  • Using a Lyse and wash procedure on peripheral blood samples enables the use of the two Scatter parameters, FSC and SSC to define the 3 major cell populations in blood. Medium FSC, low SSC define the lymphocyte population, High FSC medium SSC, defines the monocyte population, and Medium FSC and high SSC defines the Granulocyte population of blood cells.
  • FIG. 4: Using one parameter (label) to contra-select for two different populations of entities
  • The same label is used on CD14 and CD15 antibodies (Qdot800), to contra-select the monocytes and the granulocytes from the analysis of the lymphocyte population. This illustrate an example of using a single label molecule to label more than one marker molecule, for the simultaneously, contra selection of more than one defined population using only one parameter.
  • FIG. 5: Spectral overlap between the label FITC into the detector dedicated to measure RPE
  • The emission spectra of a fluorochrome label (e.g. FITC) may overlap with the spectra of another fluorochrome label (e.g. RPE). When present on an entity at the same time (e.g. CD3/FITC and CD4/RPE on lymphocytes), some of the signal from one label is detected and added to the signal of the other label measured in the detector for the second fluorochrome (CD3/RPE). To determine the signal coming from the RPE label only, subtraction of the signal from the other label has to be performed. It is therefore needed to know how much the signal from one label (FITC) is detected in the detector of the other label (RPE dedicated detector) (as illustrated). The slope of the straight line is the spillover coefficient for FITC into the RPE dedicated detector: (101-39.8)/(225-91.4) ˜45% of the FITC signal measured in the FITC dedicated detector is added to the signal measured in the RPE dedicated detector. This coefficient can be determined for all fluorochromes using single stained samples, or mixed samples where each entity only is labeled with one fluorochrome. Using linear algebra on the intensity measured on each entity the spillover can be subtracted from each signal, a process called compensation.
  • FIG. 6: A basic lymphocyte phenotyping
  • Peripheral blood was assayed. The data shown are selected for the lymphocyte population using scatter parameters, and contra-selection for monocytes and granulocytes as illustrated in FIG. 4.
  • The following detections molecules are, as shown: anti-CD3/FITC antibody, anti-CD56/RPE antibody, anti-CD4/Qdot705™ antibody, anti-CD8/Qdot800™ antibody, anti-CD20/APC antibody, anti-CD19/Qdot605™ antibody, and anti-CD5/Qdot655™ antibody. Spillover has been determined between all labels, as illustrated in FIG. 5, and the data has been compensated for that spillover for each cell, and plotted. The intensity scale of each label (detection molecule) is shown on the axes.
  • FIG. 7: Example of exclusion gating
  • The data shown illustrate the use of exclusion gating. Entities that are defined as having very low FSC and very low SSC, are marked on the scatter plot, and cells being all cells not defined by this region is the cells of interest. This is an example of how to use parameters to reduce the amount of data, entity measurements that are saved in the file.
  • FIG. 8: Counter selection of cells using a DNA stain
  • The sample was stained with 7-AAD, a DNA stain that can not enter into viable cells but can enter necrotic and dead cells. Thus viable cells will be negative, while necrotic and dead cells will be positive for this detection molecule. The viable cell data can now be further processed and/or the amount of dead cells present in the sample calculated. This is also an example of a label and marker being the same molecule.
  • FIG. 9: Detection of antigen specific T cells using MHC multimer constructs simultaneously with activation and intracellular staining of cytokines.
  • The figures illustrate IFN-γ versus Pentamer staining of live lymphocytes I a blood sample from a human donor. PBMCs were incubated with either a negative control (non-specific) Pentamer (A*0201/EBV (GLCTLVAML)) or a Pentamer specific for the cells of interest (B*0801/EBV (RAKFKQLL)), then stimulated with LAC (non-specific activation) or B*0801/EBV peptide (specific peptide activation) for 15 hours in the presence of Brefeldin A. Fixation, permeabilization and staining for IFN-γ were carried out exactly as detailed in the protocol in example 55.
  • FIG. 10: Detection of Borrelia specific T cells in sample from human donor.
  • Human peripheral blood lymphocytes were ficoll purified and stained with either of a pool of MHC/APC dextramer molecule constructs containing peptides derived from Borrelia antigen Osp A and FlaB or the cells were stained with the negative control construct HLA-A*0201/GLAGDVSAV/APC. Both samples were also stained with the antibodies mouse-anti-human CD3/PB (clone UCHT1 from Dako), mouse-anti-human CD4/FITC (clone MT310 from Dako) and mouse-anti-human CD8/PE (clone DK25 from Dako). Following staining the sample was analyzed on a CyAn flow cytometer. Live-gated, CD4 negative and CD3 positive lymphocytes are shown. Dot plots showing live gated CD3+/CD4− lymphocytes from Borrelia patient stained with (A) Negative Control MHC Dextramer (HLA-A*0201(GLAGDVSAV) or (B) pool of MHC Dextramers containing peptides from Borrelia antigen Osp A and Fla B pool of MHC Dextramers containing peptides from Borrelia antigen are shown.
  • 0.05% of the live gated CD3+/CD4− lymphocytes are positive for one or more of the MHC Dextramers in the pool.
  • FIG. 11: HLA-typing using HLA-specific antibodies.
  • Human peripheral blood lymphocytes were ficoll purified from two donors, donor 1 and donor 2. Samples of cells from each donor were stained with PE-labeled anti-HLA-A*02 and anti-HLA-A*03 antibodies respectively and then analyzed on a flow cytometer. As a control unstained cell samples from each donor was also analyzed. Cells were gated using a lymphocyte gate in a FCS/SSC plot and the presence of PE positive staining was determined in each sample. As shown by the histogram plots in the figure donor 1 was positive for HLA-A*02 and negative for HLA-A*03 and donor 2 was negative for HLA-A*02 and positive for HLA-A*03.
  • FIG. 12: Gating strategy for no-lyse no-wash procedure.
  • Whole blood was stained with MHC multimer, anti-CD8/APC, anti-CD3/PB and CD45/CY antibody in a no-lyse no-wash procedure. For further details see text in example 37. During analysis of data the following gating strategy was used: CD45/PB antibody was used to set a trigger discriminator to allow the flow cytometer to distinguish between red blood cells and stained white blood cells. This was done during data collection by gating on CD45/PB positive cells in a CD45/PB vs. side scatter dot plot as shown in A. After data collection and during data analysis CD3 positive cells were selected by gating CD3/FITC positive cells in a CD3/FITC vs side scatter plot as shown in B. The final data was illustrated in a MHC multimer/PE vs CD8/APC plot (see FIG. 13).
  • FIG. 13: Identification of CMV-specific T cells in a blood sample using no-lyse no-wash procedure
  • Whole blood from three different donors were analyzed for the presence of CMV-specific T cells by flow cytometry using a no-lyse no-wash procedure. Donor 1 was stained with a MHC multimer consisting of PE-conjugated 270 kDa dextran coupled with HLA-A*0201 in complex with beta2microglobulin and the peptide NLVPMVATV derived from Human Cytomegalo Virus (HCMV) (left panel) and with a negative control MHC multimer consisting of PE conjugated 270 kDa dextran coupled with HLA-A*0201 in complex with beta2microglobulin and the peptide ILKEPVHGV derived from Human Immunodeficiency Virus (HIV) (right panel). Donor 2 was stained with a MHC multimer consisting of PE-conjugated 270 kDa dextran coupled with HLA-A*0101 in complex with beta2microglobulin and the peptide VTEHDTLLY derived from Human Cytomegalo Virus (HCMV) (left panel) and a negative control MHC multimer consisting of PE-conjugated 270 kDa dextran coupled with HLA-A*0101 in complex with beta2microglobulin and the peptide IVDCLTEMY derived from ubiquitin specific peptidase 9 (USP9) (right panel). Donor 3 was stained with two MHC multimers consisting of PE conjugated 270 kDa dextran coupled with HLA-B*0207 in complex with beta2microglobulin and either of the peptides TPRVTGGGAM (left panel) or RPHERNGFTVL (center panel) both derived from Human Cytomegalo Virus (HCMV) and with a negative control MHC multimer consisting of PE-conjugated 270 kDa dextran coupled with HLA-B*0207 in complex with beta2microglobulin and the peptide TPGPGVRYPL derived from Human Immunodeficiency Virus (HIV) (right panel). All samples were also added Anti-CD45/PB, anti-CD3/FITC and anti-CD8/APC antibodies. The samples were gated as shown in FIG. 12.
  • FIG. 14: Enumeration of specific T cells using CytoCount™ beads.
  • Whole blood from a human donor were analyzed for the presence of CMV-specific T cells with MHC multimers by flow cytometry using a no-lyse no-wash procedure. 2×100 μl donor blood was analyzed with two different MHC multimers: A) PE-conjugated 270 kDa dextran coupled with HLA-A*0101 in complex with beta2microglobulin and the peptide VTEHDTLLY derived from Human Cytomegalo Virus (HCMV) and a negative control construct B) consisting of PE-conjugated 270 kDa dextran coupled with HLA-A*0101 in complex with beta2microglobulin and the peptide IVDCLTEMY derived from ubiquitin specific peptidase 9 (USP9). To each sample Anti-CD45/CY, anti-CD3/APC and anti-CD8/PB antibody was added together with 50 μl CytoCount beads (1028 beads/μl). Following staining for 15 minutes PBS was added to 1 ml and the samples analyzed on a CyAn flow cytometer. During analysis CD45/CY antibody was used to set a trigger discriminator to allow the flow cytometer to distinguish between red blood cells and stained white blood cells and CD3/APC antibody was used to gate for CD3 positive T lymphocytes.
  • Amount of counted beads in sample A are shown in the histogram C and amount of beads counted in the negative control sample B are show in histogram D.
  • Concentration of HLA-A*0101(VTEHDTLLY) specific T cells in the blood sample was determined as follows:

  • ((count of MHC multimer+CD8+cells in A×concentration of beads×dilution factor of beads)/counted beads C))−((counted MHC multimer+CD8+cells in B×concentration of beads×dilution factor of beads)/counted beads D)=((1300 cells×1028 beads/μl×0.05)/67225 beads)−((2 cells×1028 beads/μl×0.05)/72623 beads)=0.9926 cells/=μl=992.6 celler/ml
  • FIG. 15: Flow chart showing the process of measurement of antigen reactive T-Cells by IFN-γ capture in blood samples by ELISPOT assay.
  • See example 20 for description of process steps in ELISPOT assay.
    •  DEFINITIONS
  • Antibodies: As used herein, the term “antibody” means an isolated or recombinant binding agent that comprises the necessary variable region sequences to specifically bind an antigenic epitope. Therefore, an antibody is any form of antibody or fragment thereof that exhibits the desired biological activity, e.g., binding the specific target antigen. Antibodies can derive from multiple species. For example, antibodies include rodent (such as mouse and rat), rabbit, sheep, camel, and human antibodies. Antibodies can also include chimeric antibodies, which join variable regions from one species to constant regions from another species. Likewise, antibodies can be humanized, that is constructed by recombinant DNA technology to produce immunoglobulins which have human framework regions from one species combined with complementarity determining regions (CDR's) from a another species' immunoglobulin. The antibody can be monoclonal or polyclonal. Antibodies can be divided into isotypes (IgA, IgG, IgM, IgD, IgE, IgG1, IgG2, IgG3, IgG4, IgA1, IgA2, IgM1, IgM2)
  • Antibodies: In another embodiment the term “antibody” refers to an intact antibody, or a fragment of an antibody that competes with the intact antibody for antigen binding. In certain embodiments, antibody fragments are produced by recombinant DNA techniques. In certain embodiments, antibody fragments are produced by enzymatic or chemical cleavage of intact antibodies. Exemplary antibody fragments include, but are not limited to, Fab, Fab′, F(ab′)2, Fv, and scFv. Exemplary antibody fragments also include, but are not limited to, domain antibodies, nanobodies, minibodies ((scFv-CH3)2), maxibodies ((scFv-CH2—CH3)2), diabodies (noncovalent dimer of scFv).
  • Antigen: Substance that cause an immune response
  • Antigen presenting cell: An antigen-presenting cell (APC) as used herein is a cell that displays foreign antigen complexed with MHC on its surface.
  • Antigenic peptide: Any peptide molecule that is bound or able to bind into the binding groove of either MHC class 1 or MHC class 2.
  • CMV: Cytomegalo Virus
  • Counting beads: Beads or particles that can be used as internal control beads enabling absolute cell count in a sample.
  • Detection molecule: A molecule or a complex comprising a marker molecule and a labeling molecule. A detection molecule can be both in one molecule.
  • Hapten; a residue on a molecule for which there is a specific molecule that can bind, such as an antibody. Fluorescein, in addition of being a fluorochrome, it may also work as a hapten, for which there is an antibody that can bind specifically.
  • Labeling molecule: Used interchangeably with label herein. A molecule that can be detected in an assay. Example labeling molecules are chromophores (e.g. coomassie blue), fluorochromes (e.g. FITC), radioactive molecules (e.g. molecules containing the radionuclide P-32 or S-35). Precipitated molecules exposing a hapten.
  • Marker molecule: Used interchangeably with marker herein. A molecule that specifically associates covalently or non-covalently with a molecule belonging to or associated with an entity.
  • MHC: Denotes the major histocompatibility complex.
  • A “MHC Class I molecule” as used everywhere herein is defined as a molecule which comprises 1-3 subunits, including a heavy chain, a heavy chain combined with a light chain (beta2m), a heavy chain combined with a light chain (beta2m) through a flexible linker, a heavy chain combined with a peptide, a heavy chain combined with a peptide through a flexible linker, a heavy chain/beta2m dimer combined with a peptide, and a heavy chain/beta2m dimer with a peptide through a flexible linker to the heavy or light chain. The MHC molecule chain can be changed by substitution of single or by cohorts of native amino acids or by inserts, or deletions to enhance or impair the functions attributed to said molecule. By example, it has been shown that substitution of XX with YY in position nn of human beta2m enhance the biochemical stability of MHC Class I molecule complexes and thus can lead to more efficient antigen presentation of subdominant peptide epitopes.
  • MHC complex: MHC complex is herein used interchangeably with MHC-peptide complex, unless it is specified that the MHC complex is empty, i.e. is not complexed with peptide.
  • A “MHC Class II molecule” as used everywhere herein is defined as a molecule which comprises 2-3 subunits including an alpha-chain and a beta-chain (alpha/beta-dimer), an alpha/beta dimer with a peptide, and an alpha/beta dimer combined with a peptide through a flexible linker to the alpha or beta chain, an alpha/beta dimer combined through an interaction by affinity tags e.g. jun-fos, an alpha/beta dimer combined through an interaction by affinity tags e.g. jun-fos and further combined with a peptide through a flexible linker to the alpha or beta chain. The MHC molecule chains can be changed by substitution of single or by cohorts of native amino acids or by inserts, or deletions to enhance or impair the functions attributed to said molecule. Under circumstances where the alpha-chain and beta-chain have been fused, to form one subunit, the “MHC Class II molecule” can comprise only 1 subunit.
  • “MHC complexes” and “MHC constructs” are used interchangably herein.
  • “MHC protein” and “MHC molecule” are used interchangably herein. Accordingly, a functional MHC peptide complex comprises a MHC protein or MHC molecule associated with a peptide to be presented for cells or binding partners having an affinity for said peptide.
  • By the terms “MHC complexes” and “MHC multimers” as used herein are meant such complexes and multimers thereof, which are capable of performing at least one of the functions attributed to said complex or multimer. The terms include both classical and non-classical MHC complexes. The meaning of “classical” and “non-classical” in connection with MHC complexes is well known to the person skilled in the art. Non-classical MHC complexes are subgroups of MHC-like complexes. The term “MHC complex” includes MHC Class I molecules, MHC Class II molecules, as well as MHC-like molecules (both Class I and Class II), including the subgroup non-classical MHC Class I and Class II molecules.
  • The MHC molecule can suitably be a vertebrate MHC molecule such as a human, a mouse, a rat, a porcine, a bovine or an avian MHC molecule. Such MHC complexes from different species have different names. E.g. in humans, MHC complexes are denoted HLA. The person skilled in the art will readily know the name of the MHC complexes from various species.
  • MHC dextramers: MHC-peptide complexes coupled to dextran through covalent or non-covalent interactions. The dextramer can furthermore comprise flourochrome or other label coupled to dextran molecules through covalent or non-covalent interactions.
  • MHC multimer: The terms MHC multimer, MHCmer and MHC'mer herein are used interchangeably, to denote a complex comprising more than one MHC-peptide complexes, held together by covalent or non-covalent bonds.
  • MHC molecule: Herein denotes the empty MHC protein, i.e. MHC protein not complexed with antigenic peptide.
  • MHC multimer: The terms MHC multimer, MHCmer and MHC'mer herein are used interchangeably, to denote a complex comprising more than one MHC-peptide complexes, held together by covalent or non-covalent bonds.
  • MHC tetramer: MHC tetramers are MHC multimers consisting of streptavidin/avidin associated with 4 biotinylated MHC molecules.
  • Negative control peptide: peptide binding the MHC allele of choice, but that does not support binding of the resultant MHC-peptide complex to the desired TCR
  • RBC: Red Blood cell
  • TB: Tuberculosis
  • Vacutainer: a registered brand of test tube specifically designed for venipuncture. The tube has vacuum inside and a rubber cap in the top. The vacuum causes blood to move through the needle inserted into the rubber cap and into the tube.
  • The term “entity” as used herein refers to any cell, molecule, molecular complex, particle or sub-component, detectable or selectable by the use of marker molecules, and specific inherent characteristics. Example entities are cells such as T-cells or B-cells, cell like particles, micelles and liposome's, beads, particles, polymer beads, supramolecular structures and supramolecular compartments.
    • DETAILED DESCRIPTION OF THE INVENTION
  • The present invention in one embodiment is directed to the analysis and sorting (partitioning) of entities such as cells, supramolecular compartments and particles. The analytical principles can include binding of the entities to e.g. affinity columns or other types of solid supports, flow cytometry or stationary cytometry, such as immunohistochemistry.
  • In one embodiment, the entities to be assayed and/or sorted are MHC molecules. Alternatively, the MHC molecules can be used in the assaying and/or sorting of the entities in question. The MHC molecules can be MHC multimers as disclosed in more detail herein below.
  • In another embodiment, the entities to be assayed are T cells or antigen presenting cells. Alternatively, the T cells and/or antigen presenting cells can be used in the assaying. Antigen presenting cells are herein any cell expressing MHC I molecules and/or MHC II molecules.
  • The present invention also includes analysis of substances released from entities e.g. soluble factors secreted from cells (cytokines, hormones ect.)
  • MHC molecules function as antigenic peptide receptors, collecting peptides inside the cell and transporting them to the cell surface, where the MHC-peptide complex may be recognized by T-lymphocytes. Two classes of classical MHC molecules exist, MHC class I and II. The most important difference between these two molecules lies in the protein source from which they obtain their associated peptides. MHC class I molecules present peptides derived from endogenous antigens degraded in the cytosol and are thus able to display fragments of viral proteins and unique proteins derived from cancerous cells. Almost all nucleated cells express MHC class I on there surface even though the expression level varies among different cell types. MHC class II molecules bind peptides derived from exogenous antigens. Exogenous proteins enter the cells by endocytosis or phagocytosis and these proteins are degraded by proteases in acidified intracellular vesicles before presentation by MHC class II molecules. MHC class II molecules are only expressed on professional antigen presenting cells like B cells and macrophages.
  • The three-dimensional structure of MHC class I and II molecules are very similar but important differences exist. MHC class I molecules consist of two polypeptide chains, a heavy chain, a, spanning the membrane and a light chain, β2-microglobulin (β2m). The heavy chain is encoded in the gene complex termed the major histocompatibility complex (MHC), and comprises three domains, α1, α2 and α3. The β2m chain is not encoded in the MHC gene and consists of a single domain, which together with the α3 domain of the heavy chain make up a folded structure that closely resembles that of the immunoglobulins. The α1 and α2 domains pair to form the peptide binding cleft, consisting of two segmented a helices lying on a sheet of eight 3-strands. In humans as well as in mice three different types of MHC class I molecule exist. HLA-A, B, C are found in humans while MHC class I molecules in mice are designated H-2K, H-2D and H-2L.
  • The MHC class II molecule is composed of two membrane spanning polypeptide chains, α and β, of similar size (about 30000 Da). Genes located in the major histocompatibility complex encode both chains. Each chain consists of two domains, where α1 and β1 forms a 9-pocket peptide-binding cleft, where pocket 1, 4, 6 and 9 are considered as major peptide binding pockets. The α2 and β2, like the α2 and β2m in the MHC class I molecules, have amino acid sequence and structural similarities to immunoglobulin constant domains. In contrast to MHC class I molecules, where the ends of the peptide is buried, peptide-ends in MHC class II molecules are not. HLA-DR, DQ and DP are the human class II molecules, H-2A, M and E are those of the mice.
  • A remarkable feature of MHC genes is their polymorphism accomplished by multiple alleles at each gene. The polygenic and polymorphic nature of MHC genes is reflected in the peptide-binding cleft so that different MHC molecules bind different sets of peptides. The variable amino acids in the peptide binding cleft form pockets where the amino acid side chains of the bound peptide can be buried. This permits a specific variant of MHC to bind some peptides better than others.
  • MHC Multimers
  • Due to the short half-life of the peptide-MHC-T cell receptor ternary complex (typically between 10 and 25 seconds) it is difficult to label specific T cells with labelled MHC-peptide complexes. In order to circumvent this problem, MHC multimers have been developed. These are complexes that include multiple copies of MHC-peptide complexes, providing these complexes with an increased affinity and half-life of interaction, compared to that of the monomer MHC-peptide complex. The multiple copies of MHC-peptide complexes are attached, covalently or non-covalently, to a multimerization domain. Known examples of such MHC multimers include the following:
      • MHC-dimers: Two copies of MHC-peptide; IgG is used as multimerization domain, and one of the domains of the MHC protein is covalently linked to IgG.
      • MHC-tetramers: Four copies of MHC-peptide, each of which are biotinylated, are held together in a complex by the streptavidin tetramer protein, providing a non-covalent linkage between a streptavidin monomer and the MHC protein. Tetramers are described in U.S. Pat. No. 5,635,363.
      • MHC pentamers: Five copies of MHC-peptide complexes are multimerized by a self-assembling coiled-coil domain. Oligomeric MHC multimers are described in the US 2004/209295
      • MHC dextramers: A large number of MHC-peptide complexes, typically more than ten, are attached to a dextran polymer. MHC-dextramers are described in the patent application WO 02/072631.
  • Design, Generation and Use of MHC Multimers
  • As used in the description of this invention, the term “MHC multimers” will be used interchangeably with the terms MHC'mers and MHCmers, and will include any number, (larger than one) of MHC-peptide complexes, held together in a large complex by covalent or non-covalent interactions between a multimerization domain and one or more MHC-peptide complexes, and will also include the monomeric form of the MHC-peptide complex, i.e. a MHC-peptide complex that is not attached to a multimerization domain.
  • MHC multimers thus include MHC-dimers, MHC-tetramers, MHC-pentamers, MHC-hexamers, as well as organic molecules, cells, membranes, polymers and particles that display two or more MHC-peptide complexes. Example organic molecule-based multimers include functionalized cyclic structures such as benzene rings; example cell-based MHC multimers include dendritic cells and antigen presenting cells (APCs); example membrane-based MHC multimers include liposomes and micelles carrying MHC-peptide complexes in their membranes; and example polymer-based MHC multimers include MHC-dextramers (dextran to which a number of MHC-peptide complexes are covalently or non-covalently attached). Obviously, any kind of multimerization domain may be used, including any kind of cell, polymer, protein or other molecular structure, or particles and solid supports.
  • Different approaches to the generation of various types of MHC multimers are described in U.S. Pat. No. 5,635,363 (Altmann et al.), patent application WO 02/072631 A2 (Winther et al.), patent application WO 99/42597, US patent 2004209295, U.S. Pat. No. 5,635,363, and is described elsewhere in the present patent application as well. In brief, the MHC multimers may be generated by first expressing and purifying the peptide components of the MHC protein, and then combining the MHC peptide components and the peptide, to form the MHC-peptide complex. Then an appropriate number of MHC-peptide complexes are linked together by covalent or non-covalent bonds to a multimerization domain. This may be done by chemical reactions between reactive groups of the multimerization domain (e.g. vinyl sulfone functionalities on a dextran polymer) and reactive groups on the MHC protein (e.g. amino groups on the protein surface), or by non-covalent interaction between a part of the MHC protein (e.g. a biotinylated peptide component) and the multimerization domain (e.g. four binding sites for biotin on the strepavidin tetrameric protein). As an alternative, the MHC multimer may be formed by the non-covalent association of amino acid helices fused to one component of the MHC protein, to form a pentameric MHC multimer, held together by five helices in a coiled-coil structure making up the multimerization domain.
  • The MHC multimers may be labeled with fluorochromes and used in flow cytometry. MHC multimers may be used to label T-cells carrying specific T-cell receptors (TCR). For IHC, the MHC multimers may be labeled with fluorochromes or enzymes, in order to specifically stain specific T-cells carrying TCR's that specifically bind the MHC multimer in question. ELISA and ELISA-like analyses may be performed with MHC multimers that are labeled with enzymes, chromophors or fluorochromes.
  • For all of the above applications, it is important to choose the right MHC allele as well as a peptide that binds well to the MHC protein. It is also important that the chosen MHC allele and peptide forms a MHC-peptide complex that is efficiently and specifically recognized by the TCR. For applications that involve binding as well as activation of cells, further restrictions on the choice of MHC and peptide may apply.
  • Marker Molecules
  • Marker molecules are molecules that specifically interact with target molecules on the surface or in the interior of entities such as cells or beads. Marker molecules can be any kind of molecule, for example antibodies, nucleic acids aptamers, small organic molecules and many other types of molecules. Marker molecules that are used in the analysis of cell populations include molecules that specifically bind to extra-cellular membrane proteins or associated molecules (e.g. receptors and glycosides), intracellular proteins and RNA, and DNA in the nucleus of eukaryotic cells. Particularly interesting marker molecules include so-called MHC-multimers. MHC-multimers are complexes comprising multiple MHC-peptide complexes.
  • Flow Cytometry Methods
  • Flowcytometry is a powerful method for which to analyze many entities for multiple parameters at the same time. The principal of detecting a specific molecule in or on the surface of the entity is to have a specific marker molecule, such as an antibody recognizing the molecule in question. For detection of the binding, the marker molecule is labeled with a fluorochrome, thus if the entity displays the specific target it is detected after laser light excitation by the flow cytometer as being fluorescing, and the amount of fluorescent light is proportional to the amount of marker molecules on the entity. Many such pairs of markers and target molecules linked fluorochromes can be detected at the same time by flow cytometry. The data are analyzed and interpret by selectively selecting the entities displaying certain fluorescents markers and or selecting entities lacking selective markers, a process called positive and negative or dump gating, respectively.
  • As an example, the identification of antigen specific cytotoxic T-cells are identified by the binging of specific MHC labeled molecules and that of labeled anti-CD8 antibody, as well as negative selected for the presence of CD4 identified by a labeled anti-CD4 antibody. The same process can be performed on a cell sorter, where the cells that fulfill the gating criteria is physically sorted into a purified population of cells with the phenotype described by the gating strategy. Extending the analysis to encompass more marker molecules for a more precise phenotyping of the entities of interests, will need more labeling molecule, or maybe even automated help in the gating strategy as the gating becomes increasingly more requiring with more markers involved. The full workflow from sample source and sampling to interpretation of the data is involving many different steps, which open possibility for mistakes, thus an easy and comprehensive methodology throughout the workflow is essential for reproducibility of data and reliable diagnosis of diseases.
  • Immunohistochemistry (IHC) Methods
  • Is a method to detect specific molecules in tissue sections. Sections of fixed or frozen tissues are incubated with the marker molecule, labeled with a fluorochrome, chromophore, or another molecule that may be detected. As an example, the MHC multimer may be labeled with a tag that can be recognized by e.g. a secondary antibody, optionally labeled with HRP or another label. The bound marker molecule is then detected by its fluorescence (for fluorochrome), or by addition of an enzyme-labeled antibody directed against the marker molecule, or another component of the molecule (e.g. one of the protein chains, a label on the multimerization domain, a chemical “tag or hapten”). The enzyme may be Horse Raddish Peroxidase (HRP) or Alkaline Phosphatase (AP), both of which may catalyze a chemical conversion of a non-detectable to a precipitated detectable molecule such as a precipitation of a chromophore detectable by it absorption, or by the enzymatic generation of chemiluminescence. Or it may catalyze a precipitation of molecules exposing a tag or hapten for binding of additional detection molecule in situ.
  • The colored deposit, or a label molecule bound to the exposed tag in the precipitate, identifies the binding site of the marker molecule, and can be visualized by photometry, such as general microscopy, fluorescence microscopy or digital scanning and photographic techniques.
  • The marker molecule, e.g. a MHC multimer can also be directly labeled with e.g. HRP or AP, or a fluorochrome, and used in IHC without an additional secondary binding molecule.
  • In IHC the tissue is often fixated in formalin and embedded in paraffin, thus a deparafination step using organic solvents are used before staining.
  • Antibodies are standard reagents used for staining of formalin-fixed tissue sections; some antibodies recognize linear epitopes and binds easily to fixed tissue, however other antibodies recognize conformal epitopes, or hidden epitopes in the fixed tissue, therefore, often an antigen retrieval procedure is applied, e.g heating the sample in a microwave oven, or other ways to shift the fixed conformation of the antigen. MHC multimers are expected to recognize a conformational epitope on the TCR, thus a gently fixation or a antigen retrieval procedure might be required in most cases. Alternatively, staining is performed on frozen tissue sections, and the fixation is done after MHC multimer staining.
  • Preferred Method Steps of the Present Invention
  • An analytical or preparative analysis or enrichment of a collection of entities, such as biological cells, involves a number of sub-processes or steps, such as
  • A) sampling (acquiring the sample),
  • B) sample preparation (manipulating the sample in order to obtain the best possible result in the sub-processes that follow),
  • C) assaying the sample (thereby obtaining a quantization of different kinds of entities/selection of entities with specific characteristics), and optionally
  • D) data acquisition and/or sample processing, such as partitioning or sorting of preferred entities, as well as further optionally
  • E) further processing of the obtained data set and/or F) further processing of the partitioned or sorted entities.
  • The below table aims to provide an overview of the methods of the present invention. Steps A, B and C are essential steps in so far as all samples are prepared for assaying and subsequently assayed, whereas steps D, E and F are optional and depends on the purpose and nature of the assaying method (or step) performed in (step) C.
  •
    Figure US20100248257A1-20100930-C00001
  • In order to obtain the best possible result, it is important to optimize each of the sub-processes A, B and C, as well as D, E and F (when present), with a desired result in mind.
  • The combination of different sub-processes (steps) is also crucial for the outcome of the experiments, and different starting points (i.e. sample source and sampling process, cf. step A above)) often necessitate different sample preparation steps (c.f. step B above) and the different analytical procedures subsequently applied (cf. step C, and optionally further steps D, E and F).
  • In accordance with the present invention, the analytical procedures preferably comprise method steps for analyzing and isolating entities by means of marker molecules, such as MHC molecules and MHC multimers, that are capable of binding specific target ligands present in or exposed on the entities of interest. Target ligands can be labelled.
  • The types of analyses which can be carried out in accordance with the present invention include cytometry (flow cytometry and stationary cytometry) and batch treatment. The analytical procedures (i.e. assaying step C) described herein below in more detail allow the analysis (quantization) or selection of entities, based on the characteristics of the individual entity. Thus, characteristics of the entities can include e.g. the type of entity (e.g. is the entity a T-cell or a B-cell), as well as status of the entity (e.g. is the entity a yeast cell in phase G1, G2 or S; is the cell dead or viable).
  • Below, each of the sub-processes (steps A to F) is described. Preferred sub-processes are indicated and several preferred combinations of sub-processes are described in detail.
  • Sampling (A)
  • Sampling is the process by which the sample is collected from the source and optionally kept until the sample can be prepared and analyzed.
  • Sample sources include samples obtained from live as well as non-live sources, including but not limited to humans, animals, birds, insects, plants, algae, fungi's, yeast, viruses, bacteria and phages, multi-cellular and mono-cellular organisms, and chemical reactions comprising e.g. supra-molecular structures, transitions state molecules, chemical, and enzymatic reactions. Human and animal samples include blood, semen, and tissue samples. Sources of pathogens, such as bacteria, viruses and other infective organisms and sources may be environmental samples such as drinking water, sewage, or soil.
  • Blood is a preferred sample source for flow cytometry. Other preferred human sources for flow cytometry include bodily fluids such as urine, saliva, lymphatic fluid, cerebrospinal liquid and semen, as well as solid tissue samples. In order to use tissue for flow cytometry, the tissue may be subjected to enzymatic digestion, or the tissue may be mechanically grinded or micro-dissected to yield suspensions suitable for flow cytometry.
  • For stationary cytometry such as immunohistochemistry, solid samples may be fixed and paraffin embedded, or frozen and analyzed as thin sections on slides. Liquid samples may be subjected to cytospin or immobilized by some other method on slides, prior to stationary cytometry analysis. Animal samples may be subjected to similar procedures.
  • The sample may be collected into a container that is appropriate for the intended type of analysis, or that protects the sample from the environment or protects the experimenter from the sample. The sampling devise preferably is a sterile, single-use devise, into which the sample passively drips or is actively drawn by e.g. vacuum. For blood transfusion such devises may hold up to one liter or more of blood; for analytical purposes as little as a few milliliters or less may be collected. Even smaller volumes may be obtained through micro-puncture followed by aspiration of e.g. a single drop of blood directly into the analytical devise. For patients that require frequent sample extraction, the sampling devise is preferably designed so as to allow sampling by non-professional health workers or the patient. Alternatively, the sample is drawn directly from patient circulation, e.g. during surgery, which may allow simultaneous analysis in a continuous manner.
  • Other features of sample containers include mechanical strength to allow centrifugation, transparent walls or opaque walls protecting the sample from light with transparent windows to allow visual sample monitoring. For blood sampling, the sampling needle should be easily extractable preferably directly into disposal containers. A chemical or electronic monitoring devise that integrates time and temperature following sample introduction into the devise may be used as a measure of sample integrity.
  • Preferably, the sample is collected into a container under circumstances where the volume can be easily determined or standardized; for this purpose a vacutainer is particularly preferable, as they are easy of use, and draw a specific quantity of blood. Other preferred containers include, open blood glass, donor blood bag, and capillary tubes.
  • The format of the sample container is chosen in accordance with the further analysis. Example containers include microtiter plates, sample tubes for large, e.g. 1-20 ml, or small, e.g. 10 μl-1 ml sample volume, and may be handled individually or in a dedicated rack system.
  • The sampling devise may contain anti-oxidants, anti-coagulants, biocides or other reagents prior to the addition of the sample. These reagents serve to protect the sample from the surroundings, or alternatively, protect the surroundings (e.g. the experimenter) from the sample. Example reagents are anti-coagulants such as citrate, EDTA or heparin; fixatives such as formaldehyde or glutardialdehyde; various preservatives such as azide, benzoate and or biocides against e.g. HIV virus or other viruses, or against bacteria. The reagents thus serve to maintain sample integrity until it can be prepared for, analyzed. Should the sample for practical or logistic reasons need prolonged storage prior to analyses, addition of reagents such as glycerol or DMSO will allow the sample device to be stored at below freezing temperatures for an extended period of time. Additional preservatives, such as concentrated sugar solutions, may be included to extend the shelf life of such pre-charged sampling devises. Finally, the sampling devise may contain reagents that are employed during the analytical step (e.g. marker molecules such as MHC-dextramers, antibodies, fluorescent beads or other probes and reference material to be used for the subsequent sample analyses). Alternatively, the reagents may be added during sample preparation (see below).
  • The sampling devise may also comprise mechanical devises such as filters, additional inlets and outlets that allow reagents to be added and samples extracted; solutions that by centrifugation establish a density gradient, and other features that are part of the sample preparation process. As an example, the sample may be homogenized or filtered, or may be solubilized by addition of e.g. proteases and nucleases, in order to perform e.g. a flow cytometry analysis.
  • For the downstream quantitative measurement it is preferable that the primary sample volume and concentration of added reagents are accurately known. Internal control reagents such as e.g. beads, optionally encoded and/or of known quantity and/or signal intensity (e.g. fluorescence intensity), may be added to the primary sample or to the sampling devise prior to sampling. These internal control reagents may serve as a means for quantitation of specific entities, analyzed volume of sample, effect of sample preparation and storage, or fixation (if performed) on the sample, as a standard for the assay performed, e.g. fluorescent intensity standard in cytometry. Furthermore, beads added to the sample could serve as a mean for identification of the sample at a later stage, as a measure of sample loss during the sample preparation or analysis steps that follow. Beads may be encoded by their fluorescence, either by the bead's intensity and/or by its fluorescence or multiple fluorescence's , or by its physical properties such as forward or side scatter in fluorescence-based flow cytometry. Alternatively, RFID (Radio Frequency IDentification), graphical or optical labels may be used to encode the beads. When used for identification purposes, the bead encoding scheme should ideally establish an unequivocal information link between sample origin, e.g. patient identity, time of extraction and identity of the health professional who acquired the sample.
  • Preferably, many steps of the sampling process are automated, as this reduces risk of error and minimizes the risk of accidental exposure of the experimenter to potentially infectious or in other ways hazardous materials. Ideally, the sample is transferred to a sampling devise, containing reagents that ensure sample stability and allow efficient sample preparation, where said sampling devise fits directly into a fully automated instrument for data acquisition.
  • The sample may be applied to the analytical instrument manually, or by the use of a robotic or automated device, that handles tubes from a rack and loads the sample on the instrument. Other handling devices may be used, such as a tube carrousel, tubes in an X/Y pattern rack or linear rack, and microtiter-, or multiwell plates, depending on the application and instrument used.
  • Sample Preparation (B)
  • As described above, samples may be collected from diverse sources such as human blood or tissue, environmental samples or chemical reactions. Often, additional steps must be performed in order to run the analysis on the sample. This process is called sample preparation, and include i) addition of anti-oxidants, anti-coagulants, biocides or other reagents that protect the sample from the surroundings, or alternatively, protect the surroundings (e.g. the experimenter) from the sample, ii) physical or chemical removal or destruction of troublesome contaminants, iii) addition of chemicals or processing steps that provide the sample in a form (e.g. solid or fluid) appropriate for the analytical step that follows, iv) enrichment of the desired entities, v) labeling of the entities, and vi) addition of internal standard reagents.
  • Preparation of the sample for subsequent analysis can be an integrated part of the total analysis, because the way the sample preparation is performed will influence the basic of the subsequent analysis. Sample preparation may remove or add factors, and therefore influences the analytical data acquired.
  • During sample preparation, the sample may be fractionated to enrich for or remove certain factors or entities. This may be done by physical or chemical separation. Example physical separations are simple centrifugation, density centrifugation in ficoll, glycerol, sucrose or other chemicals that alter the physical density of the sample during centrifugation. Centrifugation is often used on blood to separate different cell types; typical cell types that may be separated include red blood cells (RBC), and lymphocyte from mono- and granulocytes. An example chemical treatment is induced precipitation or destruction of contaminants or undesired entities by an added chemical.
  • For blood sample preparation, chemical separation include selective destruction of entities, e.g. lysis of RBC in full blood using chemical reagents, such as UtiLyse™, EasyLyse™, FACSLyse™, or other ammonium containing solutions followed by osmotic shock.
  • Contaminants may be removed by binding to immobilized marker molecules such as antibodies, or subpopulations of entities may be enriched by binding a sub-population of entities to solid support, for example carrying immobilized marker molecules. This type of enrichment can be used to remove undesired entities, in which case the solid support with the immobilized entities is simply discarded, or it can be used to isolate a subpopulation containing the desired entities, in which case the non-bound entities are first removed, and then the immobilized entities released from solid support.
  • Internal standard reagents may be added during sample preparation. As an example, quantitation such as determination of concentration, labeling efficiency, signal intensity or other characteristics of specific entities is often more accurate when employing an added, internal standard reagent. Example internal standard reagents include: i) a known number of particles (for quantization of amount of entities), ii) particles with known fluorescence intensity, and iii) particles capable of associating with a specific amount of a labeling dye.
  • In order to correct for increases or decreases in sample volume during sample preparation, a known number of internal control beads may be added to a known volume of the primary sample initially. This will allow for a correction for loss of material during sample preparation after the analysis, provided that the numbers of internal control beads are determined during the analytical step. Such beads are in the following referred to as counting beads. In general, the counting beads are microparticles with scatter properties that put them in the context of the cells of interest when registered by a flow cytometer. They can be either labelled with antibodies, fluorochromes or other marker molecules or they may be unlabelled. In some embodiments of the invention, the beads are polystyrene beads with molecules embedded in the polymer that are fluorescent in most channels of the flow-cytometer. Inhere the term counting bead and microparticle are used interchangeable. The counting beads employed in the methods and compositions described here should preferably be small, and are preferably between 0.1 μm and 100 μm, preferably about 5 μm in diameter. The microparticles should preferably be made of such material and be of such size as to stay suspended, with minimal agitation if necessary, in solution or suspension (i.e., once the sample is added). It should preferably not settle any faster than the cells of interest in the sample. The material from which the microparticles are made should be such as to avoid clumping or aggregation, i.e., the formation of doublets, triplets, quadruplets and other multiplets. Generally, a final count of counting beads of at least 1000/μl is preferred but any number can be counted. The counting beads should preferably be labelled with a reporter molecule, such as a fluorescent molecule (which is described in further detail elsewhere). Alternatively, or in addition, a microparticle which is autofluorescent may be employed.
  • Counting beads may be selected from the group consisting of fixed chicken red blood cells, coumarin beads, liposomes containing a fluorescent dye, fluorescein beads, rhodamine beads, fixed fluorescent cells, fluorescent cell nuclei, microorganisms and other beads tagged with a fluorescent dye. However, particularly advantageous examples of compact particles that may be used in the invention include microbeads, such as agarose beads, polyacrylamide beads, polystyrene beads, silica gel beads, etc.
  • Beads or microparticles suitable for use include those which are used for gel chromatography, for example, gel filtration media such as Sephadex. Suitable microbeads of this sort include Sephadex G-10 having a bead size of 40-120μ (Sigma Aldrich catalogue number 27, 103-9), Sephadex G-15 having a bead size of 40-120 μm (Sigma Aldrich catalogue number 27, 104-7), Sephadex G-25 having a bead size of 20-50 μm (Sigma Aldrich catalogue number 27, 106-3), Sephadex G-25 having a bead size of 20-80 μm (Sigma Aldrich catalogue number 27, 107-1), Sephadex G-25 having a bead size of 50-150 μm (Sigma Aldrich catalogue number 27,109-8), Sephadex G-25 having a bead size of 100-300 μm (Sigma Aldrich catalogue number 27, 110-1), Sephadex G-50 having a bead size of 20-50 μm (Sigma Aldrich catalogue number 27, 112-8), Sephadex G-50 having a bead size of 20-80 μm (Sigma Aldrich catalogue number 27, 113-6), Sephadex G-50 having a bead size of 50-150 μm (Sigma Aldrich catalogue number 27, 114-4), Sephadex G-50 having a bead size of 100-300 μm (Sigma Aldrich catalogue number 27, 115-2), Sephadex G-75 having a bead size of 20-50 μm (Sigma Aldrich catalogue number 27, 116-0), Sephadex G-75 having a bead size of 40-120 μm (Sigma Aldrich catalogue number 27, 117-9), Sephadex G-100 having a bead size of 20-50 μm (Sigma Aldrich catalogue number 27, 118-7), Sephadex G-100 having a bead size of 40-120 μm (Sigma Aldrich catalogue number 27, 119-5), Sephadex G-150 having a bead size of 40-120 μm (Sigma Aldrich catalogue number 27, 121-7), and Sephadex G-200 having a bead size of 40-120 μm (Sigma Aldrich catalogue number 27, 123-3).
  • Sepharose beads, for example, as used in liquid chromatography, may also be used. Examples are Q-Sepharose, S-Sepharose and SP-Sepharose beads, available for example from Amersham Biosciences Europe GmbH (Freiburg, Germany) as Q Sepharose XL (catalogue number 17-5072-01), Q Sepharose XL (catalogue number 17-5072-04), Q Sepharose XL (catalogue number 17-5072-60), SP Sepharose XL (catalogue number 17-5073-01), SP Sepharose XL (catalogue number 17-5073-04) and SP Sepharose XL (catalogue number 117-5073-60) etc.
  • Other preferred particles for use in the methods and compositions described here comprise plastic microbeads. While plastic microbeads are usually solid, they may also be hollow inside and could be vesicles and other microcarriers. They do not have to be perfect spheres in order to function in the methods described here. Plastic materials such as polystyrene, polyacrylamide and other latex materials may be employed for fabricating the beads, but other plastic materials such as polyvinyl chloride, polypropylene and the like may also be used. Polystyrene is a preferred material. The microparticles include unlabelled beads, beads with antibodies, fluorochromes or other small molecules conjugated to the surface or beads with fluorochromes embedded in the polymer.
  • Likewise, internal control reagents with known characteristics, e.g. known fluorescence intensity, allow correction of e.g. an instrument's drift as regards fluorescence measurement.
  • During sample preparation, marker molecules, optionally linked to labeling molecules, may be added. Alternatively, marker molecules may be added first, and allowed to bind the entities first, and then labeling molecules that associate with the marker molecules may be added. Entities that bind these marker molecules in this way become labeled (stained), which will allow their identification and/or isolation in the subsequent analysis step. The marker molecules may be added manually or by an automated pipetting robot. Marker molecules and labeling are described in further detail elsewhere herein.
  • Likewise, the other reagents may be added to the sample as an optimized mix of different marker molecules. Using the “Matrix” reagents system, the optimized combination and amount of reagents are immobilized in a matrix of sugars in a container, such as a tube or well of a microtiter plate. Then the sample (e.g. “untreated full blood”, diluted blood, or “lyse no wash”, or “lyse washed” blood) is added into the container and mixed with the immobilized content of the container.
  • Cells in a collected sample may be stimulated to secrete soluble factors, express new and/or increased levels of surface receptors and/or stimulated to proliferate. Cells can be stimulated by addition of antigen and/or antigenic peptide and/or addition of cytokines. Cells may also be stimulated by addition of antigen presenting cells either alone or in combination with either of the above describes stimulators. Alternatively, cells in a sample can be stimulated by addition of antibody, antibody fragments, protein ligands or other molecules whos interaction with specific receptors on the surface of the cell result in stimulation. Other stimulators include but is not limited to Lipopolysaccharide, phytohaemagglutinin (PHA), Con A, Pokeweed mitogen, other mitogens, chemicals, small organic molecules or any other substances able to stimulate cells.
  • Sample preparation for stationary cytometry, is here exemplified by a typical sample preparation process for paraffin embedded samples for use in immunohistochemistry Tissue is remove from the donor or patient, and the tissue is immediately fixated in formalin. After fixation the tissue is embedded in paraffin, which allow for storage of the tissue for years.
  • Sample preparation starts by cutting thin sections of the paraffin block, an mount the sections onto glass plates. The sample is deparafinated by incubation in organic solvents, and optionally stained with different chemical dyes, staining different compartments of the cells, e.g nucleus and the lipid membrane. Optionally, an antigen retrieval procedure is required for subsequent binding of certain antibodies. The sample can now be stained with a detection molecule, e.g. an antibody or other marker molecules linked to a fluorochrome or an enzyme for its detection. Alternatively, the sample is incubated with a marker molecule that is labeled in a subsequent process by an anti-antibody or another marker molecule linked to a label molecule, such as an HRP, or AP (Horse raddish peroxidase, or alkaline phosphatase).
  • After washing the sample to reduce unspecific staining, the sample is assayed using an appropriate method for the label used, such as by photometric analysis, e.g. a colorimetric assay where the label is an enzyme, or by fluorescence where the label is a fluorochrome, or by a combination of these techniques.
  • “Acquisition” of data is made by phase contrast or fluorescence microscopy, or by digital scanning, and the data interpret.
  • Marker Molecules, Labeling Molecules and Detection Molecules
  • Marker molecules are molecules or complexes of molecules that bind to other molecules. Marker molecules thus may bind to molecules on entities, including the desired entities as well as undesired entities. Labeling molecules are molecules that may be detected in a certain analysis, i.e. the labeling molecules provide a signal detectable by the used method. Marker molecules, linked to labeling molecules, constitute detection molecules. Sometimes a marker molecule in itself provides a detectable signal, wherefore attachment to a labeling molecule is not necessary.
  • Marker Molecules
  • Marker molecules are typically antibodies, directed against human blood cells, or other antigens as exemplified in FIG. 1, however may be directed against antigens from any organisms, e.g. mouse, yeast, or bacteria. Antibodies may be monoclonal or polyclonal and derived from any species e.g. man, mouse, rabbit, pig. The antigens are most often proteins but may also be other molecules, e.g. sugars, DNA, RNA, or small organic molecules.
  • Alternatively, marker molecules maybe, aptamers, proteins, peptides, small organic molecules, natural compounds (e.g. steroids), non-peptide polymers, MHC multimers (including MHC-dextramers, MHC-tetramers, MHC-pentamers and other MHC-multimers), or any other molecules that specifically and efficiently bind to other molecules are also marker molecules, as exemplified in Table 1. Typically, marker molecules bind molecules associated with an entity inhere defined as the target for the marker molecule.
  • Examples of marker molecules and corresponding target molecules are given below, and in FIG. 1 and Table 1.
      • Antibodies binding to membrane components on, or within cells; e.g. Polysaccharides, proteins, or lipid residues.
      • MHC multimers (e.g. MHC dextramers, MHC tetramers, MHC pentamers), complexed with a specific peptide, binds to the T-cell receptor (TCR) on T-cells.
      • MHC multimers (e.g. MHC dextramers, MHC tetramers, MHC pentamers), complexed with a so-called nonsense peptide (i.e. a peptide that binds the MHC protein but expectably does not mediate efficient MHC complex-TCR interaction with any T-cell), are used as negative control for the specific binding of a specific MHC multimers to the cell.
      • Marker molecules such as Propidium Iodide (PI) that stain DNA in cells, here the marker and the label is the same molecule.
      • Marker molecules that stain DNA, and that is used to characterize the state of a cell (e.g. state of cell cycle), e.g. Drag 5, PI, or other DNA binding molecules.
      • Markers that bind specifically to incorporated molecules. An example is BrdU, which may be added to a cell which will incorporate BrdU into its DNA; by using anti-BrdU antibody, cells that have incorporated BrdU will be detected.
      • Marker molecules such as hormones, or growth factors, that specific interacts with a cellular component, such as the estrogen receptor with estrogen or the EGF receptor with EGF.
      • Introduction of modified (and optionally labeled) nucleotides, amino acids, or vitamins into cells which incorporate these into cellular components that subsequently can be detected, by themselves (e.g. if they are radioactive, or fluoresce) or by their association with a detection molecule.
  • Table 1: Example, Marker Molecules and Description of Example Targets, and Example Diagnostic Value.
  • The table Lists examples of marker molecules, being antibodies or other molecules, what the marker binds, what sort of molecule the marker is and the value of different marker groups is, which may be used in contra or pro selection in an identification assay. Thus may be used as pro or contra selective assay's as described in this invention.
  • TABLE 1
    Name Type of Molecule Target Diagnostic value
    Her2 FISH probe aptamer NEU, or HER2 Brest cancer cell gene
    DNA amplification
    TOP2A FISH probe aptamer TOP2A (DNA) proliferating cells, e.g.
    malignant cells,
    cancer cells gene
    amplification
    anti-CD8 antibodies Antibody/protein CD8 T-Cell CTL (cytotoxic
    receptor lymphocyte)
    anti-CD4 antibodies Antibody/protein CD4 T cell receptor T-helper cells
    Phosphor tyrosine Antibody/protein Tyrosine activated cells, e.g.
    specific antibody phosphorylated cancer cells
    proteins
    Phosphor Serine Antibody/protein Serine activated cells, e.g.
    specific antibody phosphorylated cancer cells
    proteins
    Estrogen Small organic molecule, ER (estrogen Cells, including Brest
    steroid receptor) cancer cells
    Morphine small organic molecule Opioid receptor neural cells
    MHC dextramer protein HLA-complex, T- Antigen specific T
    cell receptor cells
    EGF (epidermal Protein EGF receptor Brest cancer cells
    growth factor)
    Activated MEK Intracellular protein MAPKinase Growing cells
    kinase signaling molecule
    serine and or tyrosine
    kinase
    Calcium (Ca2+) Ion Calcium binding Mammalian cells, e.g.
    protein, e.g muscle cells
    calmodulin
    Anti-Phosphor serin, Phosphorylated Protein Activated cellular Cancer cells, growing
    threonine and signaling cells
    thyrosin, antibodies molecules
    Specific protein Peptide other proteins, e.g. Cells expressing
    binding domains, the RAF protein target protein, “growth
    such as RAS's RAF activated cells”
    binding domain.
    Anti-BrdU antibodies Protein/antibody BrdU incorporated Cell growth analysis
    into cellular DNA
    DNA fluorescing Small organic molecules DNA Cells containing DNA
    dyes e.g.
    Propidium iodide
    Hoechst stain
    DAPI
    AMC
    DraQ5 ™
    Acridine orange
    7-AAD
    Dead cell DNA 7-AAD DNA Dead cells
    fluorescing marker Propidium iodide
    Only dead cells are
    penetrable for these
    dyes
  • Marker molecules may bind to the surface of an entity (e.g. membrane proteins of a cell), or may bind to the interior of the entity (e.g. molecular structures within a cell, such as proteins, membranes, or nucleic acids).
  • Labeling Molecules
  • Labeling molecules are molecules that are detectable in a specific assay, and the amount of labeling molecule may be quantified.
  • Different principles of labeling and detection exist, based on the specific property of the labeling molecule, including molecules that absorb, excite, or modify radiation, such as emission of radioactive radiation (radionuclide, isotopes), absorption of light (e.g. dyes, chromophores), emission of light after excitation (fluorescence from fluorochromes), nuclear magnetic resonance form paramagnetic molecules (NMR), reflection of light (scatter from e.g. such as gold-, plastic- or glass-beads/particles of various sizes and or shapes).
  • Furthermore, labelling molecules may have an enzymatic activity, by which it catalyze a reaction between chemicals in the near environment of the labeling molecules, producing a signal which include production of light (chemi-luminescence) or precipitation of chromophors, dyes, or a precipitate that can be detected by an additional layer of detection molecules. The enzymatic product may deposit at the location of the enzyme, in cell based assays, react with the membrane of the cell, or diffuse into the cell to which the enzymatic marker is attached, in this way labeling the cell.
  • A single labelling molecule on a marker does not always generate sufficient signal intensity. The signal intensity may be improved by assembling single label molecules into large multi-labelling compounds, containing two or more label molecule residues.
  • Generation of multi-label compounds may be achieved by covalent or non covalent, association of labelling molecules with a major structural molecule. Such structures may be synthetic or natural polymers e.g. dextrans, DNA, PNA, a protein such as streptavidin. The label molecules in a multi-labelling compound may all be of the same type or may be a mix of different labelling molecules.
  • Table 2: Example Labeling Substances and Example Detection Principle.
  • The table lists labelling substances that may be used as labeling molecules in any of the described assays. Also is described how the label “gives the signal”, and the physical appearance of the signal, as well as the detection principle employed, may any of these labels be used in the herein described methods.
  • TABLE 2
    Labeling substance Effect Assay-principle
    Fluorochromes e.g. Table 4a emission of light having a
    Figure US20100248257A1-20100930-P00001
    Photometry, Microscopy,
    and b specific spectra spectroscopy
    PMT, photographic film,
    COD's (Color-Capture Device
    or Charge-coupled device).
    Radionuclide irradiation, α, β or γrays Scintillation counting, GM-
    tube, photographic film,
    excitation of phosphor-
    imager screen
    Enzyme; catalysis of H2O2 reduction
    Figure US20100248257A1-20100930-P00001
    Photometry, Microscopy,
    HRP, horse raddish using luminol as Oxygen spectroscopy
    peroxidase, acceptor, resulting in oxidized PMT, photographic film,
    peroxidases in general luminal + light CCD's (Color-Capture Device
    catalysis of H2O2 reduction or Charge-coupled device),
    using a soluble dye, or Secondary label linked
    molecule containing a hapten, antibody
    such as a biotin residue as
    Oxygen acceptor, resulting in
    precipitation. The habten can
    be recognized by a detection
    molecule.
    Particles; gold, polystyrene Change of scatter, reflection Microscopy, cytometry,
    beads, pollen and other and transparency of the electron microscopy
    particles associated entity PMT's, light detecting
    devices, flowcytometry scatter
    Alkaline Phosphatase Catalyze a chemical
    Figure US20100248257A1-20100930-P00001
    Photometry, Microscopy,
    conversion of a non- spectroscopy
    detectable to a precipitated Secondary label linked
    detectable molecule, such as antibody
    a dye or a hapten
    Ionophores or chelating Change in absorption and
    Figure US20100248257A1-20100930-P00001
    Photometry, Cytometry,
    chemical compounds binding emission spectrums when spectroscopy
    to specific ions, e.g. Ca2+ binding.
    Change in intensity
    Lanthanides Fluorescence
    Figure US20100248257A1-20100930-P00001
    photometry, cytometry,
    Phosphorescence spectroscopy
    Paramagnetic NMR (Nuclear magnetic
    resonance)
    DNA fluorescing stains Propidium iodide
    Figure US20100248257A1-20100930-P00001
    Photometry, cytometry,
    Hoechst stain spectroscopy
    DAPI
    AMC
    DraQ5 ™
    Acridine orange
    7-AAD
    Figure US20100248257A1-20100930-P00001
    Photometry; being any means that can be applied to detect the intensity, analyze the wavelength spectra, and or measure the accumulation of light coming form a source emitting light of one or multiple wavelength or spectra.
  • Table 3a: Example fluorochromes. Example fluorochromes which may be used to label any of the marker molecules described in this invention, and the maximum excitation and the emission wavelengths for these fluorochromes.
  • Excitation Emission
    Fluorofor/Fluorochrome nm nm
    2-(4′-maleimidylanilino)naphthalene-6-sulfonic 322 417
    acid, sodium salt
    5-((((2-iodoacetyl)amino)ethyl)amino) 336 490
    naphthalene-1-sulfonic acid
    Pyrene-1-butanoic acid 340 376
    AlexaFluor 350 (7-amino-6-sulfonic acid-4- 346 442
    methyl coumarin-3-acetic acid
    AMCA (7-amino-4-methyl coumarin-3-acetic 353 442
    acid
    7-hydroxy-4-methyl coumarin-3-acetic acid 360 455
    Marina Blue (6,8-difluoro-7-hydroxy-4-methyl 362 459
    coumarin-3-acetic acid
    7-dimethylamino-coumarin-4-acetic acid 370 459
    Fluorescamin-N-butyl amine adduct 380 464
    7-hydroxy-coumarine-3-carboxylic acid 386 448
    CascadeBlue (pyrene-trisulphonic acid acetyl 396 410
    azide
    Cascade Yellow 409 558
    Pacific Blue (6,8 difluoro-7-hydroxy coumarin- 416 451
    3-carboxylic acid
    7-diethylamino-coumarin-3-carboxylic acid 420 468
    N-(((4-azidobenzoyl)amino)ethyl)-4-amino-3,6- 426 534
    disulfo-1,8-naphthalimide, dipotassium salt
    Alexa Fluor 430 434 539
    3-perylenedodecanoic acid 440 448
    8-hydroxypyrene-1,3,6-trisulfonic acid, 454 511
    trisodium salt
    12-(N-(7-nitrobenz-2-oxa-1,3-diazol-4- 467 536
    yl)amino)dodecanoic acid
    N,N′-dimethyl-N-(iodoacetyl)-N′-(7-nitrobenz-2- 478 541
    oxa-1,3-diazol-4-yl)ethylenediamine
    Oregon Green 488 (difluoro carboxy 488 518
    fluorescein)
    5-iodoacetamidofluorescein 492 515
    propidium iodide-DNA adduct 493 636
    Carboxy fluorescein 495 519
  • Table 3b: Example Preferable Fluorochrome Families.
  • Many fluorochrome families have been generated by alterations of commonly known fluorochromes, and some new principles for fluorescence labeling has been generated by dedicated companies as well, e.g. Qdot™'s. This table shows examples off preferable fluorochrome families that can be obtained and used directly in the methods described for detection in this invention.
  • Fluorochrome family Example fluorochrome
    AlexaFluor ®(AF) AF ® 350, AF405, AF430, AF488, AF500, AF514, AF532,
    AF546, AF555, AF568, AF594, AF610, AF633, AF635,
    AF647, AF680, AF700, AF710, AF750, AF800
    Quantum Dot (Qdot ®) based Qdot ® 525, Qdot ® 565, Qdot ® 585, Qdot ® 605, Qdot ® 655,
    dyes Qdot ® 705, Qdot ® 800
    DyLight ™ Dyes (DL) DL549, DL649, DL680, DL800
    Small fluorescing dyes FITC, Pacific Blue ™, Pacific Orange ™, Cascade Yellow ™,
    Marina blue ™, DSred, DSred-2, 7-AAD, TO-Pro-3,
    Cy-Dyes Cy2, Cy3, Cy3.5, Cy5, Cy5.5, Cy7
    Phycobili Proteins: R-Phycoerythrin (RPE), PerCP, Allophycocyanin (APC), B-
    Phycoerythrin, C-Phycocyanin
    Fluorescent Proteins (E)GFP and GFP ((enhanced) green fluorescent protein)
    derived mutant proteins; BFP, CFP, YFP, DsRed, T1, Dimer2,
    mRFP1, MBanana, mOrange, dTomato, tdTomato,
    mTangerine, mStrawberry, mCherry
    Tandem dyes with RPE RPE-Cy5, RPE-Cy5.5, RPE-Cy7, RPE-AlexaFluor ® tandem
    conjugates; RPE-Alexa610, RPE-TxRed
    Tandem dyes with APC APC-Aleca600, APC-Alexa610, APC-Alexa750, APC-Cy5,
    APC-Cy5.5
    Calcium dyes Indo-1-Ca2+ Indo-2-Ca2+
  • Different detection principles for detection of labels exist. Examples listed in Table 2, may be applied to flow cytometry, stationary cytometry, and batch-based analysis. Most batch-based approaches can use any of the labelling substances described, depending on the purpose of the assay. Flow cytometry primarily employs fluorescence, whereas stationary cytometry primarily employs light absorption, e.g. dyes or chromophor-deposit formed by enzymatic activity. Therefore, in the following section, principles involving fluorescence detection will be exemplified for flow cytometry, and principles involving chromophore detection will be exemplified in the context of stationary cytometry. However, the labelling molecules may in principle be applied to any of the analyses described in this invention.
  • The labelling molecules may be associated, linked or covalent bound to the marker molecule and added to the sample. Alternatively, the marker molecule are first allowed to associate with the specific target, then the label molecule is added, binding specifically to the marker molecule. The latter involves that the marker and the label is modified in a way that will allow a specific association between the marker and the label. This may be archived by any means that mediates a specific interaction between the marker and the labelling molecule. Means as described for the association between the maker molecule and the target, may be applied to mediated specific binding pairs of molecules, such as Antigen/Antibodies, streptavidine/biotin, nucleotides base pairing (DNA:DNA, RNA:RNA, RNA:DNA), or PNA with PNA, PNA with DNA or RNA, where the bead is attached to one of the partner molecules (e.g. the antigen) and the label is attached to the other partner molecule (e.g. the antibody).
  • Preferable Fluorescent Labeling in Flowcytometry
  • In flowcytometry the typical label is detected by its fluorescence, examples are given in Table 3. In most cases a positive detection is based on the presents of light from a single fluorochrome, but in other techniques the signal is detected by a shift in wavelength of emitted light; as in FRET based techniques, where the exited fluorochrome transfer its energy to an adjacent bound fluorochrome (and marker) that emits light, or when using Ca2+ chelating fluorescent probes, which change the emission (and absorption) spectra when binding to calcium.
  • Preferable labelling molecules employed in flowcytometry are described in the following and in Table 3a and b.
  • Simple fluorescing labels:
      • Fluor dyes, Pacific Blue™, Pacific Orange™, Cascade Yellow™,
      • AlexaFluor®(AF);
        • AF405, AF488,AF500, AF514, AF532, AF546, AF555, AF568, AF594, AF610, AF633, AF635, AF647, AF680, AF700, AF710, AF750, AF800
      • Quantum Dot based dyes, QDot® Nanocrystals (Invitrogen, MolecularProbs)
        • Qdot®525, Qdot®565, Qdot®585, Qdot®605, Qdot®655, Qdot®705, Qdot®800
      • DyLight™ Dyes (Pierce) (DL);
        • DL549, DL649, DL680, DL800
      • Fluorescein (Flu) or any derivate of that, ex. FITC (fluorescein isothiocyanate)
      • Cy-Dyes
        • Cy2, Cy3, Cy3.5, Cy5, Cy5.5, Cy7
      • Fluorescent Proteins;
        • RPE(R-phycoerythrin), PerCp, APC(Allophycocyanin); other of phycobillin containing proteins, e.g. phycobiliprotein
        • Green fluorescent proteins (GFP);
          • GFP and GFP derivated mutant proteins; BFP, CFP, YFP, DsRed, T1, Dimer2, mRFP1,MBanana, mOrange, dTomato, tdTomato, mTangerine, mStrawberry, mCherry
      • Tandem dyes:
        • RPE-Cy5, RPE-Cy5.5, RPE-Cy7, RPE-AlexaFluor® tandem conjugates; RPE-Alexa610, RPE-TxRed
        • APC-Aleca600, APC-Alexa610, APC-Alexa750, APC-Cy5, APC-Cy5.5
      • Multi fluorochrome assemblies
        • Multiple fluorochromes attached to a polymer molecule, such as a peptide/protein, Dex, or poly-sacceride.
        • Any combination of the fluorescent dyes involving in generation of FRET (Fluorescence resonance energy transfer) based techniques.
        • Multible fluorochromes associated or coupled to a polymeric molecule, or consisting of polymeric residues.
      • Ionophors; ion chelating fluorescent probes
        • Probs that change wavelength when binding a specific ion, such as Calcium
        • Probs that change intensity when binding to a specific ion, such as Calcium
      • Combinations of fluorochromes on the same marker. The marker is not identified by a single fluorochrome but by a code of identification being a specific combination of fluorochromes, as well as inter related ratio's of intensities.
        • Example: Antibody Ab1 and Ab2, is conjugated to both FL1(ex. FITC), and Fl2 (ex. PB), but Ab1 have 1 FL1 to 1 FL2, whereas Ab2 have 2 Fl1 to 1 Fl2. Each antibody may then be identified individually by the relative intensity of the two, or more fluorochromes. Any such combinations of n fluorochromes with m different ratios may be generated.
  • Choosing Fluorochrome in Multicolor Staining Protocols
  • Although the choice of fluorochrome or fluorochrome combinations on a specific marker molecules, in particular antibodies, or MHC'multimers are more or less unlimited, a few rules may be employed to give better resolution and clearer identification of the specific cell population that are assayed.
      • Fluorochromes used on specific marker molecules detecting targets that are co-located on the same cell or entity are selected to have a minimum of spectral overlap.
      • The brightest fluorochrome is used for detection of the weakest expressed molecules on the cell
      • The binding molecules and detection molecules are optimized for the ability to give the best possible separation between the positive and negative population.
      • The detection molecules are optimized for having the least binding to cell populations that are not specific interacting with the marker molecule, thus, optimized for having as little unspecific binding as possible.
  • Preferable Labelling Molecules Employed in Stationary Cytometry and IHC
      • Enzymatic labelling, as exemplified in Table 4:
        • Horse raddish peroxidase; reduces peroxides (H2O2), in the presence of an oxygen acceptor the signal are generated its oxidation.
          • Precipitating dyes; Dyes that when reduced is soluble, and precipitate when oxidized, generating a colored deposit at the site of the reaction.
          • Precipitating agent, carrying a chemical residue, a hapten, this is exposed from the precipitate, and may be used for a second layer binding of detector molecules, resulting in amplification of the primary signal.
          • Luminol reaction, generating a light signal at the site of reaction.
        • Other enzymes, such as Alkaline Phosphatase (AP), capable of converting a chemical compound from a non detectable molecule to a precipitated detectable molecule. The detectable molecule may be colored, or carrying a hapten which may be recognizable by an detections molecule, as described above.
      • Fluorescent labels, as exemplified in Table 3a and b; however, labels described for Flow cytometry may also be used in stationary cytometry, such as in fluorescent microscopy.
  • Table 4: Example Preferable Labels for Stationary Cytometry
  • Examples of labeling principles preferably used and developed for use in stationary cytometry, but may be used in other detection principles as well, such as flowcytometry.
  • The grouping describes the labels, name, what the effector molecule is, how it manifest itself in the assay, any secondary binding pair if used in a second layer of detection.
  • Manifestation of the Second layer of
    Name of label Effector molecule effector molecule detection:
    Label Enzyme substrate, Precipitate with or Binding partner to
    Oxygen acceptor without a residue, hapten
    Chromogen
    Figure US20100248257A1-20100930-P00002
    /    		 hapten* for
    precipitating agent amplification of the
    signal
    HRP diaminobenzidine Colored precipitate —
    (DAB)
    HRP 3-amino-9-ethyl- Colored precipitate —
    carbazole (AEC+)
    AP Fast red dye Red precipitate —
    HRP biotinyl tyramide Exposed Biotin residue Streptavidin, avidine
    HRP fluorescein tyramide Exposed Fluorescein Anti-Fluorecein
    residue Antibody
    “Enzyme” Substrate that when Primary label; being a Secondary label in
    reacted precipitate dye, chemiluminescence's, case the primary label
    or exposure of a hapten is a hapten
    *Hapten; being any residue/molecule that can be recognized as a binding site for another molecule which may be labled for amplification of the primary signal.
    Figure US20100248257A1-20100930-P00002
    A chromogen is a molecule that will become a dye molecule/chromophor when e.g. oxidized by the presence of the label, enzyme.
  • Combinatorial Use of Fluorochromes: Extending the Marker Panel by Labeling Different Markers Using the Same Fluorochrome(s)
  • In order to reduce the number of different labeling molecules e.g. fluorochromes in a particular flow analysis, the use of the same fluorochrome on different marker molecules can be employed.
  • Three main scenarios, A, B, C are exemplified below:
      • A. Markers labeled by same label identify two or more subpopulations of interest in a sample. Using this method the subpopulations will not be separately identified. Groups of marker molecules can be labeled with same label and other groups of marker molecules in same preparation are labeled with one or more other labels. Marker molecules identified by same label can be positively selected as a group or negatively selected as a group.
        • Example of positive selection of a group. Three markers A, B and C are necessary to identify two different subpopulations of cells in a sample. Both populations (P) can bind marker C. One binds marker A but not marker B (PA+,B−,C+,) and the other population binds marker B and not marker A (PA−,B+,C+). Hence both populations are positive for two of the three markers. If marker A and B are both labeled with label L1 and marker C is labeled with label L2, three marker reagents are labeled with only two labels (AL1, BL1 and CL2) and these three reagents can be used to detect two subpopulations that furthermore can be differentiated from the rest of the entities in the sample using the third marker. This type of labeling are outlined in the following example:
      • A human blood sample are analyzed for the presence of cytotoxic T cell populations specific for CMV or specific EBV. The sample are stained using a CMV specific MHC-dextamer and an EBV specific MHC-dextramer, both labeled with the same label 1 (e.g. RPE). The cytotoxic T cell marker CD8 is labeled with label 2 (e.g. FITC), and used to separate the cytotoxic T cell population from the rest of the cells in the sample. The label 2 labeled CD8 positive cell population are then analyzed for the presence of CMV and/or EBV specific cytotoxic T cells using the two specific MHC-Dextramers labeled with label 1
        • Example negative selection of a group. Markers identified by the same fluorochromes are being negatively selected for, thus the final population of interest is negative for markers identified by the same fluorochrome. For example a sample contains three populations of entities identified by the markers A, B and C where only the population defined by marker C is of interest. Marker A and B are then labeled with the same and deselected during the gating strategy. Entities labeled with marker C can then be identified among the remaining entities. This kind of negative selection is especially useful to “clean up” samples for unwanted entities prior to analysis for entities of interest.
      • B. The same label can be used for two markers where one marker identifies a positive selected population and the other marker identifies a negatively selected population if either of the populations are simultaneously labeled with a third marker labeled with a different label. For example: An antigen specific cytotoxic T cell population in a human blood sample can be identified using the following combination of markers, labels and gating strategy. Anti-CD19 marker is labeled with label 2 (e.g. RPE), anti-CD3 marker is labeled with label 1 (e.g. FITC), and anti-CD8 marker is labeled with label 3 (e.g. APC). The antigen specific T cells are identified with a specific MHC-dextramer carrying a specific antigen peptide and this reagent is also labeled with label 2 (e.g. RPE), as is the CD19 marker. Cells positive for label 2 and negative for label 1 are negatively selected. The remaining cells being label 1 and label 2 positive the label 3 positive cells are selected. This group of cells will be CD19 negative, CD3 positive, CD8 positive and MHC-dextramer positive and are the antigen presenting cytotoxic T cells analyzed for.
      • C. Markers labeled with the same label can identify and distinguish two or more sub populations of entities, if each marker define a population that is also positive for an additional marker the other population(s) is/are negative for. For example 6 different marker molecules (A, B, C, D, E, F) are labeled using 3 different labels L1, L2, L3 as follows: Marker molecule A, B and E are labeled with L1, marker molecule C, D and E are labeled with L2 and marker molecule B, C and F are labeled with L3. Thereby all 6 marker molecules can be distinguished from each other by one or more labels: Marker molecule A is labeled with L1, marker molecule B is labeled with L1 and L3, Marker molecule C is labeled with L2 and L3, Marker molecule D is labeled with L2, marker molecule E is labeled with label L1 and L2 and marker molecule F is labeled with label L3
  • Assaying (C)
  • As described above, the sample can be either solid or a fluid suspension. If the sample is fluid it may be applied to the analytical instrument by positive or negative pressure applied to the sample. In flowcytometry, the sample is a suspension of entities, which are moved to and centered in the flow cell (interrogation point) by co-flow with sheath fluid, or is directly injected into the instrument. If the sample is a solid it is typically placed in the instrument in the form of a thin slide of material on a glass plate. Solid materials may be processed physical and/or enzymatically into a suspension, or assayed directly without further manipulation.
  • In the present invention, analysis shall involve the use of marker molecules that associate with specific entities, and may also involve inherent physical characteristics measured by the instrument employed e.g. scatter parameters in flowcytometry. By these parameters the detection of specific entities are allowed. Likewise, selection (also termed enrichment, isolation or sorting) shall involve the use of marker molecules that associate with certain entities, and may as well involve inherent physical characteristics that can be detected by the technique employed.
  • Example entities are cells and cell like particles, micelles and liposome's, polymer beads, and supramolecular structures, or any other molecular complex or particle or sub-component, detectable or selectable by the use of marker molecules, and specific inherent characteristics.
  • Analyses, as described in the present invention, can in principle be divided into two different categories: Cytometry and batch-based analysis. Cytometry may be further divided into flow cytometry and stationary cytometry. The following introduces analytical principles applied in the various analysis.
  • Cytometry
  • Cytometry allows detection and investigation of physical and/or chemical properties of each entity in the sample analyzed. Cytometry may be grouped into flow cytometry (e.g., fluorescence-activated cell sorting (FACS)) and stationary cytometry (e.g. immunohistochemistry (IHC)). In flow cytometry the sample is in a liquid form, a suspension of entities which is flowing through an interrogation-point, whereas, in stationary cytometry the sample is typically a solid.
  • Flow cytometry allows detection of a single entity with a specified set of characteristics, within a population of entities with other sets of characteristics. A major advantage of flowcytometry is that it allows rapid analysis of multiple parameters for each individual entity, simultaneously.
  • In the most widely used flow cytometry instruments, such as the CyAn ADP™ (Dako), FACS-Calibur, and Canto (BD biosciences), or the EPIC and FC 500 (Beckmann Coulter), the sample is transported to the flow cell by applying pressure to the sample tube and focused in the flow cell using sheath fluid. A sheath fluidic system accelerates each entity into the flow-cell resulting in a focused line of entities. As a result, the entities pass through the interrogation point one at a time.
  • Alternatively, in micro-fluidics-based, flow-on-a-chip system (a “dry” flow cytometer), the sample is passed through the interrogation point by the means of micrometer-scale tubing's, using capillary forces acting on the liquid in a capillary tube.
  • Labelling molecules can be molecules that absorp, excite, or modify radiation, e.g light, and radioactivity. Obviously, the detection methods reflect this.
  • In flow cytometry, the most commonly used detection parameters are the scatter and fluorescence parameters, but other means of detection, e.g. color (absorption) and radioactivity may be used as well. The scatter parameters are defined as the quantity of light detected in a forward (FSC) and a sideward or 90° scatter (SSC), or in any other defined angle to the light source and the sample stream. These measurements provide information on characteristics such as relative size, transparency and granularity of the entity. The fluorescence parameters describe the fluorescence that is emitted from the detection molecules associated with the entity. One or more lasers of well defined wavelengths are used to generate scatter light from, and to excite the fluorochromes associated with, each entity. A common combination of lasers is the 405 nm-, 488 nm- and 635 nm-lasers, as used in the CyAn ADP™ flowcytometer. However, the number of lasers may be more or less than that. Furthermore, lasers emitting tuneable and multiple wavelengths of light, conventional light sources or diode-based light sources can also be employed. Additionally, one may measure transmitted (absorbed), or reflected light, rather than fluorescence, from the entities.
  • When an entity or sample comprises two or more different fluorochromes, a number of principles for the detection of the individual fluorochromes may be employed. These include:
      • The photomultipliers are performing intensity measurements of light of discrete wavelength intervals. The light emitted by the fluorochromes of each entity may be split into a number of different channels, each corresponding to discrete wavelength intervals, as the light goes through an optical system comprised of multiple dichroics and optical filters, prisms, diffraction grating, or photonic crystals. Finally the quantity of light within a defined wavelength interval is measured using a PMT (photomultiplier tube)-based detection system, whereby the fluorescence intensity of each fluorochromes may be determined, thus providing a measure of the abundance of each marker molecule bound to the individual entity.
      • Total wavelength spectra intensity measurement. By performing full wavelength spectra analysis on each entity, and combining this with knowledge of the individual fluorochromes emission spectrum, one may calculate the abundance of each of the fluorochromes bound to an entity.
      • Imaging. The emitted light may also be analyzed using a photo-responsive chip, or a multi-channel photomultiplier. In this way a 2-dimensional image of the entity may be obtained. This can provide further informative data about the entity, such as cell morphology, shape and the location of bound marker molecules on the entity.
      • Fluorescence decay time measurements. Determination of the decay rate of the emitted light over time may be applied to identify the identity and abundance of each of the labeling molecules (and hence identity and abundance of marker molecules) bound to the individual entity. This requires an oscillating excitation light source, and involves first a pulse excitation of the fluorochromes attached to the entity, and then determination of the emitted light over time as the fluorescence decays. By using an appropriate mathematical model and knowledge about each of the fluorochromes' half-life and emission spectra, the abundance of each fluorochrome is determined of the fluorochrome signals. This approach has several advantages compared to traditional fluorescence intensity measurements, including better separation. As a result, more fluorochromes may be employed simultaneously in an experiment.
      • Enzymatic labeling; this principle may involves an enzyme as the labeling molecule, capable of converting a chemical compound from a solvable molecule to a precipitated product or a reactive product that reacts with molecules in the vicinity of the enzyme, the detectable molecule is associated to the entity, or entering the entity, e.g. a cell. The precipitate may be fluorescing, or carrying a hapten for a second layer of detection.
  • Stationary cytometry is primarily employed in analysis of solid samples (e.g. slices of paraffin embedded tissue or slices of frozen tissue), however also smears of liquid samples may be analyzed by this method. Probably the most common type of stationary cytometry is immunohistochemistry (IHC).
  • The assay technique employed in stationary cytometry typically involves immobilization of the tissue slice on a glass slice, carrying the sample through the assay steps to the final analysis.
  • The sample is “stained” or labeled, using principles as described for markers and labeling elsewhere herein, and in the following.
      • Precipitate labeling; this principle typically involves an enzyme as the labeling molecule, capable of converting a chemical compound from a non-detectable molecule to a precipitated detectable molecule. The precipitated molecule may be colored, or carry a hapten recognized by another detectable molecule in a second layer of detection.
      • The enzyme is attached to the marker molecule. Binding of the marker molecule to its specific target on an entity, leads to the immobilization of the enzyme on the entity. Then, a substrate is added that is turned into a precipitated product by the enzyme, if the product precipitates or reacts with molecules in the vicinity of the enzyme, the signal will accumulate in the vicinity of the immobilized enzyme. This product, may give a signal by itself, e.g. fluoresce or absorb light at a given wavelength and thus be directly detectable. However, the product may additionally, or only, carry a residue, that is recognized by another detection molecule, which may be enzyme labeled, or labeled by any means as described elsewhere within this invention.
      • The principle of enzyme-catalyzed precipitation of dyes (or molecules carrying a second target molecule for amplification of the signal) is particularly useful in stationary cytometry.
      • “Reaction” labeling; this principle typically involves an enzyme, such as peroxidase, as the labeling molecule. Additions of the substrates generate a reaction at the site of the detection molecule which emits light of a certain wavelength and spectra, which in turn can be detected by photometry.
      • The most commonly used type of labeling in stationary cytometry is based on chromophores, e.g. which by catalytic reaction with the labeling enzyme is precipitated in the vicinity of the detection molecule. However also radioactivity and fluorescence are used.
  • The means of detection, typically involve photometric methods, microcopy and/or digital scanning of the stained sample. It may be simple light or fluorescence microscopy, for determination of chemiluminescence, morphology, shape, and fluorescence. Also, laser scanning techniques may be employed, where confocal microscopy or standard light microscopy is employed to give a 3- and 2-dimensional picture, respectively, of the sample. A digital image may be acquired, whereby the individual features e.g. light intensity at a given area of the sample can be determined.
  • Batch-Based Assay
  • Under circumstances where it is not the characteristics of a given entity that is sought, but rather the determination of the total amount of entities with a given sample, or the isolation of these entities, batch-based techniques may be used. Batch based techniques are here defined as methods that capture entities using marker molecules that bind to specific target molecules on the entity. Following the captured entities may be isolated and enriched. The sample used in these assay is preferably liquid-based samples. Liquid-based samples here means samples that by nature is liquid (e.g. blood, lymph) or samples that may be liquefied prior to analysis (e.g. suspensions of solid tissue, cultured cells in suspension, beads in suspension ect.).
  • Methods for Capturing Entities and/or Further Analysis of Entities
  • In order to isolate a subpopulation of entities, specific marker molecules may be immobilized on a solid support. Such support may be any which is suited for immobilization, separation etc. Non-limiting examples include particles, beads, biodegradable particles, sheets, gels, filters, membranes (e. g. nylon membranes), fibres, capillaries, needles, microtitre strips, tubes, plates or wells, combs, pipette tips, micro arrays, chips, slides, or indeed any solid surface material. The solid or semi-solid support may be labelled, if this is desired. The support may also have scattering properties or sizes, which enable discrimination among supports of the same nature, e.g. particles of different sizes or scattering properties, colour or intensities. Then the sample is incubated with the solid support. After incubation the solid support is washed to remove unbound sample, and the entities that remain associated with the surface of the solid support are recovered. The sample is thereby enriched for entities defined by the specific marker. Following recovery the selected entities may be expanded or their number determined. Determination of the amount of entities may also be made when the entities are still on the solid support.
  • Methods for capturing entities can also be used for analysis and/or isolation of substances or entities secreted from entities in a sample or produced in the sample during preparation of the sample.
  • The marker molecules may be immobilized directly to the solid support or may be immobilized through a linker molecule. The linker molecule can be an antibody specific for the marker molecule, a peptide or a any other molecule able to bind the marker molecule. In any case the linker may be attached directly to the solid support, the linker may be attached to the solid support through another linker or the linker is embedded in a matrix, e.g. a sugar matrix.
  • Below examples of different principles for capturing of entities in a sample using marker molecule attached to solid supports are given.
      • A solid support with one type of specific marker molecules immobilized. When the sample is incubated with the solid support, specific entities are bound to the marker molecules on the support and the bound entities may subsequently be quantitated and/or recovered.
      • A solid support with different markers immobilized in a defined pattern. The amount of entities bound to the defined areas of the support may be analyzed, thereby, phenotyping the sample. Each individual entity is defined by the target molecules it expose and depending on these target molecules each entity will bind to different marker molecules immobilized at defined positions on the solid support.
      • A solid support consisting of different beads with different characteristics (e.g. different size, different fluorescence's or different fluorescence intensities), where each kind of bead has a specific type of marker molecule immobilized. The amount of entities bound to the specific populations of beads can be analyzed, thereby, phenotyping the sample. The target exposed on the entity is defined by the bead to which it binds.
  • The advantage of capture methods is that it is possible to assay large sample volumes for entities carrying target molecules specific for various markers. This is simply done by passing the sample over the solid support (e.g. a column containing beads) or by adding the solid support (e.g. beads) directly into the sample allowing the entities to bind to its specific immobilized marker molecules. If the solid support is e.g. beads and these are incubated with the sample in suspension the beads and thereby the bound entities may be isolated by centrifugation or filtration and analyzed by e.g. flowcytometry or any other method as described in this invention. Using solid support surfaces in macro size (e.g. a chip or a glass slice), the isolation is simply done by removing the support from the suspension of entities and analyzed by any method as described elsewhere herein.
  • The amount of entities associated with markers on a solid support may be analyzed while bound to the solid support or the entities may be released/recovered and analyzed or expanded and analyzed.
  • The identity and amount of captured entities may be assayed by methods involving staining with a second specific detection molecule, or by staining with a generic stain, e.g. protein, lipid, or DNA stain. In batch based methods the target molecule exposed on the entity is defined by the bead, area or solid support to which it binds. However, the captured entities may be characterized further by employing a secondary detection layer, consisting of marker molecules directed against other targets, to the captured entities. Alternatively this second detection layer may release bound entities from the solid support by competing with the immobilized marker molecules for binding to defined targets.
  • The analysis may be supplemented with inherent characteristics of the entity, i.e. characteristics that can be detected without the use of marker molecules. Examples of such characteristics include enzymatic activity in a cell, secretion of molecules from a cell, auto-fluorescence from the entity, granularity, size, morphology in general ect.
  • Techniques Employed in Detection of Entities
  • The type of instrument used for detection of various labeling techniques of course depends on the kind of label used. Fluorochromes may be detected by photometry such as a laser-based system e.g. flow cytometry or by fluorescence microscopy. Enzymatic precipitation of dye molecules or chemiluminescence's may be examined by photometry or standard light microscopy. Radio nuclides may be detected with a Geiger Muller tube like method, a phosphor-imager, by a method based on the radioactivity induced excitation of molecules in a material that can by “read off” by induced emission of light. Lanthanides may be detected by its fluorescence, phosphorescence or NMR signal.
  • Data Acquistion (D1) and Sorting of Entities (D2)
  • Once the samples has been assayed (step C) as disclosed herein above, a number of further method steps can be carried out. Examples of further, optional method steps include e.g. data acquisition (step D1) and sorting (partitioning) of the entities (D2).
  • Using the marker molecules described herein above, optionally labeled with the label molecules described above, a data set for a population of entities is obtained (for analysis); or alternatively (for the cytometry sorting process), a data set for each entity is obtained, allowing its possible isolation, or alternatively (for the batch-based sorting process), a sub-population of entities are physically isolated by e.g. immobilization to a solid support, and may optionally be released enriching a specific population of entities.
  • Data Acquisition (D1)
  • The phenotype of the individual entity is determined by its characteristic combination of properties. These can be physical properties of the entity, such as morphology of a cell, determined by immunohistochemistry, forward- and side-scatter of a cell in a flow cytometry analysis, or these can be chemical properties of the entity, such as ability of a certain cell type to bind specific marker molecules, or the level of certain ions, such as Calcium measured by ionophores.
  • For the purpose of this invention, “gating strategy” shall be used as the term describing the strategy of choosing the physical (e.g. scatter) and chemical (e.g. fluorescence from a labeled marker molecule) parameters to be used for identification of specific entities. Thus, the gating strategy is defined by the marker molecules which have been added during sampling or sample preparation, as well as by the physical properties measured during the analysis. Example gating strategies are described in Examples.
  • The gating strategy may involve a positive as well as a negative selection of sub-populations. Thus, desired entities may be labeled with one or several labels, that then define the target entities, whereas undesired entities may be labeled with one or several labels, whereby the unwanted entities are identified and may be disregarded (for analysis), dumped or deselected in a sorting process. In this way, a systematical positive and/or negative selection of populations of entities with similar properties will lead to identification and enrichment of the entities with the desired properties.
  • When analyzing or selecting for rare events (i.e. entity populations of low abundance), and/or attempting to minimize false positives and false negatives, more than one kind of marker molecule, or physical properties are typically employed. In the following an example of selecting fore rare events is given. The abundance of an antigen specific cytotoxic T-cell population in a blood sample obtained from a human being is very low, thus a gating strategy using both positive and negative selection may be employed to detect and analyze for the presence of antigen specific T cells. Cytotoxic T-cells are defined by being a lymphocyte, having low scatter properties, having the ability to bind to the anti-CD8 markers among others and not expressing CD4. The specificity of cytotoxic T cells may be determined by the ability to associate specifically with a class 1 MHC-multimer carrying a specific peptide. In a flow cytometry analysis or cell sorting experiment the sample may be stained with markers specific for CD4 and CD8 and with one ore more specific MHC multimers, e.g. MHC dextramers. The gating strategy may then be first to deselect data points/cells with high scatter, and ability to bind the anti-CD4 marker. Among the significantly reduced amount of cells, a positive selection for the ability to bind both to the anti-CD8 marker and the MHC dextramers is employed. This, despite the rare occurrence of the specific T-cells, their detection is made possible through the use of gating reagents that select or deselect for the individual cell, in this way bringing down the number of cells that must be analyzed for MHC-dextramer marker, which effectively minimizes the number of false positives.
  • It is important for the efficiency and accuracy of the analysis that the parameters (marker molecules, labeling molecules and other characteristics, e.g. side- and forward scatter) measured are chosen with consideration to amount and kind of target molecules present on the entity, as well as the degree of spectral overlap between the emploied fluorochromes, and that the markers do not interfere with other markers bound to the entity. Thus, the quality of a flow cytometry analysis depends significantly on the gating strategy chosen.
  • In a preferable embodiment, a gating strategy is applied that involves the labeling of more than one marker molecule with the same labeling molecule. This allows collective identification of more than one population of entities. If such populations are negatively selected, it has additional advantage, as these labels are not situated on the entity of interests, thus any potential interference between the detection molecules, such as spectral overlap, undesired FRET, quenching, or marker/label interference is eliminated between these. Another preferred embodiment, is therefore preferable to use many e.g. 5 negative selection marker molecules, e.g. labeled with the same labeling molecule and fewer positive selection markers, e.g. one, than using e.g. 6 positive selection markers and no negative selection markers. The consequence of the first choice is that the cells of interest only has one marker bound, where as, in the latter, 6 markers are bound to the cell of interest.
  • In the following an example is given of how grouping of markers by labeling the markers with the same label may be used to negatively select a group of entities with different characteristics. In analysis of a sample where the target entity is a cytotoxic lymphocyte (CTL), recognizing a specific class 1 MHC-peptide complex labeled with RPE, markers that bind to undesired cells (i.e. cells that do not display the TCR sought) may all be labeled with the same label, e.g. fluorescein. These markers could be anti-CD4, anti-CD14, anti-CD15, anti-CD19, and anti-CD71 antibodies, all labeled with fluorescein (FITC), as these markers will bind to the undesired T-helper cells, monocytes, granulocytes, B-lymphocyte and red blood cells (RBC), respectively. The spillover from fluorescein into the RPE channel can be neglected as fluorescein and RPE will never be situated on the same entity. The data obtained on the fluorescein labeled entities may then be disregarded or the cells may be dumped during a sorting process.
  • Identification and or analysis of a particular group or family of entities within a sample may require a number of different parameters, i.e. detections molecules, and labels, which may not be possible to detect on the instrument employed. Grouping of markers may help solved such problem.
  • Below an example illustrating how grouping of markers, by labeling the markers with the same label, may be used for positive selection of a group of entities with many different characteristics is given. Identification and analysis of specific CTL's (Cytotoxic T Lymphocytes) involved in the immune response against a family of pathogens e.g. CMV, EBV, BK viruses or Borealis, where the goal is to investigate whether there is an ongoing infection directed against the pathogen family as a whole. This may be performed using MHC multimers each labeled with a different label, where each MHC multimer reagent carries a different peptide or a different allele or combinations thereof identifying individual antigen I specific T cell clones directed against different pathogen members of the pathogen family. However, in many analyses so many different MHC multimers are needed to ensure coverage of all the different types of pathogens in the family that all of them cannot be used in the same sample. An alternatively approach is therefore to label all or subsets of the involved MHC multimer reagents with the same labeling molecule. The advantage is that it reduces the total number of markers, freeing parameters for additional analysis of other characteristics of the specific CTL, such as the activation status. Grouping of markers by using the same label on different markers is not restricted to grouping of the above mentioned markers but can be used on any group of markers. Identification of a group of entities by using the same labeling molecule on different markers may be used for positive as well as negative selection of different entities.
  • Quantization
  • It is often desirable to know the concentration or amount of specific entities, marker molecules or labeling molecules in the sample. Such quantization of cells or particles with a certain phenotype (e.g. specific T cells carrying a specific TCR) is often made easier if the sample volume is accurately determined. Accurate measurement of the sampled volume analyzed may be done by simple volumetric measurement in a syringe-based device, or by weighting the sample continually as it enters the analytical instrument. Alternatively, or in addition, a known number of beads may be added to the initial primary sample (where the primary sample volume is known), and then, after sample preparation and after analysis of the sample, the fraction of the initial primary sample that has been analyzed can be determined by determining the amount of beads that contained in the analyzed sample. Thus, as an example, if 1000 beads were added to the primary sample, and 100 beads were observed in the sample that was analyzed, it may be concluded that 10% of the primary sample was analyzed.
  • Therefore, if e.g. 10 cells of the desired kind were detected in the sample analyzed, it can be concluded that the primary sample contained 100 cells of the desired kind. Particles may be detected and counted by e.g. their fluorescence or scatter characteristics in flow cytometry, or by visual inspection in microscopy.
  • The beads may be labeled internally, by absorption of e.g. fluorescent dyes (such beads are called “hard-dyed” beads), or fluorochromes may be chemically cross-linked, or adsorbed to the bead surface. The fluorochrome can also be attached by specific binding pairs of molecules, such as Antigen/Antibodies, streptavidine/biotin, nucleotides base pairing (DNA:DNA, RNA:RNA, RNA:DNA), or PNA with PNA, PNA with DNA or RNA, where the bead is attached to one of the partner molecules (e.g. the antigen) and the label is attached to the other partner molecule (e.g. the antibody).
  • In a preferred embodiment, the relative amount of certain specific entities is desired, relative to other entities in the sample. For example, in blood samples the amount of blood plates is often relatively constant in different individuals, and therefore, the number of specific cells in a blood sample may be normalized with the measured amount of blood plates in the sample. Also the absolute count of a subpopulation of entities is desirable, such as the total amount of CD4 positive cells, or specific MHC-multimer positive cells in the blood sample of a patient.
  • Compensating for the Spectral Overlap Between Fluorochromes in Multicolor Flow Cytometry Analysis
  • When two or more fluorochromes are employed simultaneously in flow cytometry, the spectra may overlap, resulting in an error on the intensity measured in a specific wavelength interval, by light from other fluorochromes. To get the intensity specifically coming from a specific fluorochrome, this “spillover” of light may be compensated for. First the fluorescence is measured for each single fluorochrome in all channels (intervals of wavelengths) (attached to entities, e.g. beads). The spillover coefficients between detectors measuring in different wavelengths intervals are determined mathematically.
  • The spillover coefficient can be manually calculated, or can be calculated by automated software, such as Summit™ software. The spillover coefficients are used to correct the measured signal intensity for each fluorochrome in multicolor flow cytometry for spillover from the other fluorochromes. This may be performed by employing linear algebra on the intensity measured for each entity, subtracting the signals coming from the other fluorochromes from that of the primary fluorochrome for that detector, when displaying the results.
  • Digital Imaging in Stationary Cytometry
  • When using multiple labels in stationary cytometry, digital deconvolution can help distinguish different features (e.g. colors, and morphologies) of the sample. At present time using IHC, the primary goal is to define the localization of the marker molecule, and only roughly determinate the amount of marker bound defined by the label used. Using microscopy, differentiation between the labeling molecules used (e.g. the fluorochromes in fluorescence microscopy) on each marker may be performed by making overlays of exposures done with different emission filters, thus if digitalized the spillover correction can be done in the same way as described for flow cytometry.
  • Alternatively, a high resolved digitalized color image, obtained be e.g. CCD's may contain more information about the spectral properties of the emitted light (higher resolution of the wavelength data recorded), as well as much higher amount of light recorded, then does a PMT detection in flow cytometry, therefore, by knowing the spectral properties of each of the fluorochromes it is possible to deconvolute the intensity from each of the individual fluorochromes, using mathematical overlay of their spectra's.
  • Sorting (Partitioning) of Analyzed Entities (D2)
  • Analysis and sorting depend on the same data. In an analysis of a sample, data may be acquired for all the entities of the sample first, where after the data can be analyzed when the experiment is over. In sorting, this is not possible.
  • When flow cytometry is used to sort cells, a gating strategy is set up based on a small sample acquired.
  • Form these data a “sort strategy/gating strategy” is created defining a set of criteria for which each entity is analyzed. Based on this analysis in real time, a sort decision is taken; either isolate (sort out) or deselect, dumped, the entity. The flow sorter, MoFlo (Dako NS) can administrate positive sorting into four different population of entities, and dumping (or collecting) the rest.
  • In examples is shown a combination of gating criteria, that might be used to select (sort for) CTLs of a given specificity. The cell bind to the following marker molecules: MHC-dextramer with a particular specificity, anti-CD3 defining lymphocyte cells, anti-CD8 defining the CTL of which the antigen specific MHC-dex positive cells will be found, but does not bind the following marker molecules: anti-CD15, and anti CD4, which are used for counter selection of granulocytes, and T-helper cells as well as monocytes, respectively. Thus, in this example, the anti-CD4 and anti-CD15 antibodies is used as dump gate, and the MHC dextramer, anti-CD8 and anti-CD3 as positive selecting markers.
  • During sorting for rare events, there might be a drift in the measurement from the instruments. Automatic population tracking software, such as CyTrack™, may be employed during the sorting process to compensate for such drifts. The software will change the sort gate so that it always fits to the original population identified in the sample. The software is tracking changes in several dimensions, where each dimension is a parameter that is measured and recorded (e.g. fluorescence at a certain wavelength interval, and forward and side scatter).
  • Data Acquisition Assembly
  • For the analysis and sorting strategies described in this invention, both for cytometry and batch-based processes, the choice of marker molecules is critical to obtain the best possible result. The assay strategy (defined here as the combination of sample, marker and labeling molecules used in the gating strategy, and the analytical instruments employed in a particular analysis) and the choice of other reagents must be properly coordinated to achieve the optimal result. The reagents must be constructed and combined in a way so as to maximize the information output, by e.g. optimizing the label choice for each particular marker molecule, relative to the other reagents, and methods used.
  • As an example, the number and kind of markers (e.g. 10 marker reagents, A-J) must be appropriately chosen for a given task Z. The full analysis for a process involving e.g. 10 marker reagents, A-J, can thus be described as follows:
  • Ai×Bi×Ci×Di×Ei×Fi×Gi×Hi×Ii×Ji×Zi,
  • where (i) denotes a specific choice of reagent A-J, or a specific choice of task Z. I may be different or identical for A-J and Z.
  • Each of the (labeled) markers A-J may be chosen from, but not restricted to the list of markers and labels exemplified in FIG. 1 and table 1 to 3, and within this invention. The task likewise may be one of the tasks described above (e.g. sorting among different entities by flow cytometry, detection of particular cell morphologies by an immunohistochemical assay from a fixed paraffin embedded tissue sample, or enrichment of particular blood cells by adsorption to beads). If the total number of combinations of markers and labels is n, and the total number of tasks is m, then the different processes involving 10 labeled markers are:
  • A1×B1×C1×D1×E1×F1×G1×H1×I1×J1×Z1, A2×B1×C1×D1×E1×F1×G1×H1×I1×J1×Z1, . . . , An×B1×C1×D1×E1×F1×G1×H1×I1×J1×Z1, A1×B2×C1×D1×E1×F1×G1×H1×I1×J1×Z1, . . . , A1×Bn×C1×D1×E1×F1×G1×H1×I1×J1×Z1, An×Bn×C1×D1×E1×F1×G1×H1×I1×J1×Z1, . . . , . . . , . . . , An×Bn×Cn×Dn×En×Fn×Gn×Hn×In×Jn×Zm.
  • Obviously, similar assembly formulas can be described for processes involving less or more than 10 markers, and for other choices of tasks than flow cytometry.
  • Following are example assemblies of markers and labels, exemplified for flow cytometry assays.
  • Assembly 1: Identification of a specific cell population in a sample.
  • A=A detection molecule specifically binding, and identifying a cell population of interest.
  • Z=Flow cytometric analysis of the sample on any flow cytometer capable of detecting the label employed.
  • Assembly 2: Identification of a specific cell population in a sample.
  • A=A detection molecule specifically binding, and identifying a cell population of interest.
  • B=scatter parameter
  • Z=Flow cytometric analysis of the sample on any flow cytometer capable of measuring scatter parameters and the labels employed.
  • Assembly 3: Identification of a specific cell population in a sample.
  • A=A detection molecule specifically binding, and identifying a cell population of interest.
  • B=A detection molecule specifically binding to any cell population different to the cell population of interest.
  • C=scatter parameter
  • Z=Flow cytometric analysis of the sample on any flow cytometer capable of measuring scatter parameters and the labels employed.
  • Assembly 4: Identification of specific CTL's (Cytotoxic T Lymphocytes) involved in the immune response against a specific protein.
  • A=A labeled MHC-dextramer, carrying a peptide identical to a peptide sequence in a specific protein, of interest.
  • B=A detection molecule specifically binding, and identifying CTL's, where the label is different form that used on the MHC-dextramer.
  • Z=Flow cytometric analysis of the sample on any flow cytometer capable of detecting the label employed.
  • Assembly 5: Identification of specific CTL's (Cytotoxic T Lymphocytes) involved in the immune response against a specific protein.
  • A=A labeled MHC-dextramer, carrying a peptide identical to a peptide sequence in a specific protein, of interest.
  • B=A detection molecule specifically binding, and identifying CTL's, labeled with a labeling molecule different form that used on the MHC-dextramer.
  • C=A detection molecule specifically not binding to CTL's, labeled differently form those used in A and B.
  • D=scatter parameter
  • Z=Flow cytometric analysis of the sample on any flow cytometer capable of measuring scatter parameters and the labels employed.
  • Assembly 6: Identification of specific CTL's (Cytotoxic T Lymphocytes) involved in the immune response against a specific protein.
  • A=A labeled MHC-dextramer, carrying a peptide identical to a peptide sequence in a specific protein, of interest.
  • B=CD8 antibody labeled with a labeling molecule different form that used on the MHC-dextramer.
  • Z=Flow cytometric analysis of the sample on any flow cytometer capable of measuring scatter parameters and the labels employed.
  • Assembly 7: Identification of specific CTL's (Cytotoxic T Lymphocytes) involved in the immune response against a specific protein.
  • A=A labeled MHC-dextramer, carrying a peptide identical to a peptide sequence in a specific protein, of interest.
  • B=CD8 antibody labeled with a labeling molecule different form that used on the MHC-dextramer.
  • C=Scatter parameter
  • Z=Flow cytometric analysis of the sample on any flow cytometer capable of measuring scatter parameters and the labels employed.
  • Assembly 8: Identification of specific CTL's (Cytotoxic T Lymphocytes) involved in the immune response against a specific protein.
  • A=A labeled MHC-dextramer, carrying a peptide identical to a peptide sequence in a specific protein, of interest.
  • B=CD8 antibody labeled with a labeling molecule different form that used on the MHC-dextramer.
  • C=One or more than one labeled antibodies identifying cell populations different from the CTL's, all labeled with the same labeling molecule, different from that used for the MHC-dextramer, and the CD8 antibody.
  • D=Scatter parameter
  • Z=Flow cytometric analysis of the sample on any flow cytometer capable of measuring scatter parameters and the labels employed.
  • Assembly 9: Identification of specific CTL's (Cytotoxic T Lymphocytes) involved in the immune response against a specific protein.
  • A=A labeled MHC-dextramer, carrying a peptide identical to a peptide sequence in a specific protein, of interest.
  • B=CD8 antibody labeled with a labeling molecule different form that used on the MHC-dextramer.
  • C=CD3 antibody labeled with a labeling molecule different form that used on the
  • MHC-dextramer, and the CD8 antibody.
  • D=Scatter parameter
  • Z=Flow cytometric analysis of the sample on any flow cytometer capable of measuring scatter parameters and the labels employed.
  • Assembly 10: Identification of specific CTL's (Cytotoxic T Lymphocytes) involved in the immune response against a specific protein.
  • A=A labeled MHC-dextramer, carrying a peptide identical to a peptide sequence in a specific protein, of interest.
  • B=CD8 antibody labeled with a labeling molecule different from that labeling the
  • MHC-dextramer.
  • C=CD3 antibody labeled with a labeling molecule different form that used on the MHC-dextramer, and the CD8 antibody.
  • D=One labeled antibody identifying cell populations different from the CTL's, labeled with a labeling molecule different from that used for the MHC-dextramer, the anti-CD8, and anti-CD3 markers.
  • E=Scatter parameter
  • Z=Flow cytometric analysis of the sample on any flow cytometer capable of measuring scatter parameters and the labels employed.
  • Assembly 11: Identification of specific CTL's (Cytotoxic T Lymphocytes) involved in the immune response against a specific protein.
  • A=A labeled MHC-dextramer, carrying a peptide identical to a peptide sequence in a specific protein, of interest.
  • B=CD8 antibody labeled with a labeling molecule different from that labeling the MHC-dextramer.
  • C=CD3 antibody labeled with a labeling molecule different form that used on the MHC-dextramer, and the CD8 antibody.
  • D=More than one labeled antibodies, all labeled with the same labeling molecule, different from that used for the MHC-dextramer, the anti-CD8, and anti-CD3 markers.
  • E=Scatter parameter
  • Z=Flow cytometric analysis of the sample on any flow cytometer capable of measuring scatter parameters and the labels employed.
  • Assembly 12: Identification of specific CTL's (Cytotoxic T Lymphocytes) involved in the immune response against a specific protein.
  • A=A FITC MHC-dextramer, carrying a peptide identical to a peptide sequence in a specific protein, of interest.
  • B=A CD8 antibody labeled with a labeling molecule different from FITC.
  • C=One or more than one labeled antibody, all labeled with the same labeling molecule, different from FITC, and the label used for the CD8 antibody
  • E=Scatter parameter
  • Z=Flow cytometric analysis of the sample on any flow cytometer capable of measuring scatter parameters and the labels employed.
  • Assembly 13: Identification of specific CTL's (Cytotoxic T Lymphocytes) involved in the immune response against a specific protein.
  • A=A RPE MHC-dextramer, carrying a peptide identical to a peptide sequence in a specific protein, of interest.
  • B=A CD8 antibody labeled with a labeling molecule different from RPE.
  • C=One or more than one labeled antibody, labeled with the same labeling molecule, different from RPE, and the label used for the CD8 antibody.
  • E=Scatter parameter
  • Z=Flow cytometric analysis of the sample on any flow cytometer capable of measuring scatter parameters and the labels employed.
  • Assembly 14: Identification of specific CTL's (Cytotoxic T Lymphocytes) involved in the immune response against a specific protein.
  • A=A APC MHC-dextramer, carrying a peptide identical to a peptide sequence in a specific protein, of interest.
  • B=A CD8 antibody labeled with a labeling molecule different from APC.
  • C=One or more than one labeled antibody, labeled with the same labeling molecule, different from APC, and the label used for the CD8 antibody.
  • E=Scatter parameter
  • Z=Flow cytometric analysis of the sample on any flow cytometer capable of measuring scatter parameters and the labels employed.
  • Assembly 15: Identification of specific CTL's (Cytotoxic T Lymphocytes) involved in the immune response against a specific protein.
  • A=A FITC labeled MHC-dextramer, carrying a peptide identical to a peptide sequence in a specific protein, of interest.
  • B=APC labeled CD8 antibody
  • C=PB labeled CD3 antibody.
  • D=RPE labeled antibodies identifying cell populations different from the CTL's.
  • E=Scatter parameter
  • Z =Flow cytometric analysis of the sample on any flow cytometer capable of measuring scatter parameters and the labels employed.
  • Assembly 16: Identification of specific CTL's (Cytotoxic T Lymphocytes) involved in the immune response against a specific protein.
  • A=A FITC labeled MHC-dextramer, carrying a peptide identical to a peptide sequence in a specific protein, of interest.
  • B=APC labeled CD8 antibody
  • C=PB labeled CD3 antibody.
  • D=RPE labeled antibodies directed against monocytes and granulocytes, respectively.
  • E=Scatter parameter
  • Z=Flow cytometric analysis of the sample on any flow cytometer capable of measuring scatter parameters and the labels employed.
  • Assembly 17: Identification of specific CTL's (Cytotoxic T Lymphocytes) involved in the immune response against a specific protein.
  • A=A FITC labeled MHC-dextramer, carrying a peptide identical to a peptide sequence in a specific protein, of interest.
  • B=APC labeled CD8 antibody
  • C=RPE labeled CD4 antibodies
  • E=Scatter parameter
  • Z=Flow cytometric analysis of the sample on any flow cytometer capable of measuring scatter parameters and the labels employed.
  • Assembly 18: Identification of specific CTL's (Cytotoxic T Lymphocytes) involved in the immune response against a specific protein.
  • A=A FITC labeled MHC-dextramer, carrying a peptide identical to a peptide sequence in a specific protein, of interest.
  • B=APC labeled CD8 antibody
  • C=RPE labeled CD4, CD14, and CD15 antibodies
  • E=Scatter parameter
  • Z=Flow cytometric analysis of the sample on any flow cytometer capable of measuring scatter parameters and the labels employed.
  • Assembly 19: Identification of specific CTL's (Cytotoxic T Lymphocytes) involved in the immune response against a specific protein.
  • A=A FITC labeled MHC-dextramer, carrying a peptide identical to a peptide sequence in a specific protein, of interest.
  • B=APC labeled CD8 antibody
  • C=RPE labeled CD4, CD19 antibodies
  • E=Scatter parameter
  • Z=Flow cytometric analysis of the sample on any flow cytometer capable of measuring scatter parameters and the labels employed.
  • Assembly 20: Identification of specific CTL's (Cytotoxic T Lymphocytes) involved in the immune response against a specific protein.
  • A=A RPE labeled MHC-dextramer, carrying a peptide identical to a peptide sequence in a specific protein, of interest.
  • B=APC labeled CD8 antibody
  • C=PB labeled CD3 antibody.
  • D=FITC labeled antibodies identifying cell populations different from the CTL's.
  • E=Scatter parameter
  • Z=Flow cytometric analysis of the sample on any flow cytometer capable of measuring scatter parameters and the labels employed.
  • Assembly 21: Identification of specific CTL's (Cytotoxic T Lymphocytes) involved in the immune response against a specific protein.
  • A=A RPE labeled MHC-dextramer, carrying a peptide identical to a peptide sequence in a specific protein, of interest.
  • B=APC labeled CD8 antibody
  • C=PB labeled CD3 antibody.
  • D=FITC labeled antibodies directed against monocytes and granulocytes, respectively.
  • E=Scatter parameter
  • Z=Flow cytometric analysis of the sample on any flow cytometer capable of measuring scatter parameters and the labels employed.
  • 10
  • Assembly 22: Identification of specific CTL's (Cytotoxic T Lymphocytes) involved in the immune response against a specific protein.
  • A=A RPE labeled MHC-dextramer, carrying a peptide identical to a peptide sequence in a specific protein, of interest.
  • B=APC labeled CD8 antibody
  • C=FITC labeled CD4 antibodies
  • E=Scatter parameter
  • Z=Flow cytometric analysis of the sample on any flow cytometer capable of measuring scatter parameters and the labels employed.
  • Assembly 23: Identification of specific CTL's (Cytotoxic T Lymphocytes) involved in the immune response against a specific protein.
  • A=A RPE labeled MHC-dextramer, carrying a peptide identical to a peptide sequence in a specific protein, of interest.
  • B=APC labeled CD8 antibody
  • C=FITC labeled CD4, CD14, and CD15 antibodies
  • E=Scatter parameter
  • Z=Flow cytometric analysis of the sample on any flow cytometer capable of measuring scatter parameters and the labels employed.
  • Assembly 24: Identification of specific CTL's (Cytotoxic T Lymphocytes) involved in the immune response against a specific protein.
  • A=A RPE labeled MHC-dextramer, carrying a peptide identical to a peptide sequence in a specific protein, of interest.
  • B=APC labeled CD8 antibody
  • C=FITC labeled CD4, CD19 antibodies
  • E=Scatter parameter
  • Z=Flow cytometric analysis of the sample on any flow cytometer capable of measuring scatter parameters and the labels employed.
  • Assembly 25: Identification of specific CTL's (Cytotoxic T Lymphocytes) involved in the immune response against a specific protein.
  • A=A APC labeled MHC-dextramer, carrying a peptide identical to a peptide sequence in a specific protein, of interest.
  • B=RPE labeled CD8 antibody
  • C=PB labeled CD3 antibody.
  • D=FITC labeled antibodies identifying cell populations different from the CTL's.
  • E=Scatter parameter
  • Z=Flow cytometric analysis of the sample on any flow cytometer capable of measuring scatter parameters and the labels employed.
  • Assembly 26: Identification of specific CTL's (Cytotoxic T Lymphocytes) involved in the immune response against a specific protein.
  • A=A APC labeled MHC-dextramer, carrying a peptide identical to a peptide sequence in a specific protein, of interest.
  • B=RPE labeled CD8 antibody
  • C=PB labeled CD3 antibody.
  • D=FITC labeled antibodies directed against monocytes and granulocytes, respectively.
  • E=Scatter parameter
  • Z=Flow cytometric analysis of the sample on any flow cytometer capable of measuring scatter parameters and the labels employed.
  • Assembly 27: Identification of specific CTL's (Cytotoxic T Lymphocytes) involved in the immune response against a specific protein.
  • A=A APC labeled MHC-dextramer, carrying a peptide identical to a peptide sequence in a specific protein, of interest.
  • B=RPE labeled CD8 antibody
  • C=FITC labeled CD4 antibodies
  • E=Scatter parameter
  • Z=Flow cytometric analysis of the sample on any flow cytometer capable of measuring scatter parameters and the labels employed.
  • Assembly 28: Identification of specific CTL's (Cytotoxic T Lymphocytes) involved in the immune response against a specific protein.
  • A=A APV labeled MHC-dextramer, carrying a peptide identical to a peptide sequence in a specific protein, of interest.
  • B=RPE labeled CD8 antibody
  • C=FITC labeled CD4, CD14, and CD15 antibodies
  • E=Scatter parameter
  • Z=Flow cytometric analysis of the sample on any flow cytometer capable of measuring scatter parameters and the labels employed.
  • Assembly 29: Identification of specific CTL's (Cytotoxic T Lymphocytes) involved in the immune response against a specific protein.
  • A=A APC labeled MHC-dextramer, carrying a peptide identical to a peptide sequence in a specific protein, of interest.
  • B=RPE labeled CD8 antibody
  • C=FITC labeled CD4, CD19 antibodies
  • E=Scatter parameter
  • Z=Flow cytometric analysis of the sample on any flow cytometer capable of measuring scatter parameters and the labels employed.
  • Assembly 30: Identification of specific CTL's (Cytotoxic T Lymphocytes) involved in the immune response against a specific protein.
  • A=A unlabeled MHC-dextramer, carrying a peptide identical to a peptide sequence in a specific protein, of interest, and exposing a hapeten, a binding site for a specific detection molecule, e.g. the binding site on streptavidine for biotin.
  • B=A detection molecule specific for the hapten on the MHC-dextramer, such as fluorochromes linked to Biotin.
  • B=A CD8 antibody labeled with a labeling molecule different from that used in “B”.
  • C=One or more than one labeled antibody, all labeled with the same labeling molecule, different from the one used in “B”, and the label used for the CD8 antibody.
  • E=Scatter parameter
  • Z=Flow cytometric analysis of the sample on any flow cytometer capable of measuring scatter parameters and the labels employed.
  • Assembly 31: Identification of specific CTL's (Cytotoxic T Lymphocytes) involved in the immune response against a specific protein.
  • A=A unlabeled MHC-dextramer, carrying a peptide identical to a peptide sequence in a specific protein, of interest, and having free binding sites for Biotin
  • B=Multiple fluorochromes, e.g fluorescein residues linked together and linked to a biotin molecule.
  • C=APC labeled CD8 antibody
  • D=RPE labeled antibodies identifying cell populations different from the CTL's.
  • E=Scatter parameter
  • Z=Flow cytometric analysis of the sample on any flow cytometer capable of measuring scatter parameters and the labels employed.
  • Assembly 32: Identification of specific CTL's (Cytotoxic T Lymphocytes) involved in the immune response against a specific protein.
  • A=A unlabeled MHC-dextramer, carrying a peptide identical to a peptide sequence in a specific protein of interest, and having free binding sites for Biotin.
  • B=Multiple fluorochromes, e.g fluorescein residues linked together and linked to a biotin molecule.
  • C=APC labeled CD8 antibody
  • D=RPE labeled CD4, and CD19 antibodies.
  • E=Scatter parameter
  • Z=Flow cytometric analysis of the sample on any flow cytometer capable of measuring scatter parameters and the labels employed.
  • Assembly 33: Identification of specific CTL's (Cytotoxic T Lymphocytes) involved in the immune response against a specific protein.
  • A=APC labeled MHC-dextramer, carrying a peptide identical to a peptide sequence in a specific protein of interest, labled with FITC.
  • B=RPE labeled CD8, antibody
  • C=FITC labeled CD4, and CD19 antibodies.
  • D=PB labeled CD45
  • E=Scatter parameter
  • Z=Flow cytometric analysis of the sample on any flow cytometer capable of measuring scatter parameters and the labels employed.
  • Assembly 34: Identification of specific CTL's (Cytotoxic T Lymphocytes) and evaluate its activation status.
  • A=APC labeled MHC-dextramer, carrying a peptide identical to a peptide sequence in a specific protein of interest, labled with FITC.
  • B=PB labeled CD8, antibody
  • C=FITC labeled CD4, and CD19 antibodies.
  • D=Labeled CD45Ro antibody carrying a label different for the one used in A-C
  • E=Labeled CD45RA antibody carrying a label different for the one used in A-D
  • F=Labeled CD25 antibody carrying a label different for the one used in A-E,
  • G=Labeled CD27 antibody carrying a label different for the one used in A-F
  • H=Labeled CD28 antibody carrying a label different for the one used in A-G
  • I=Labeled CCR7 antibody carrying a label different for the one used in A-H
  • J=Scatter parameter
  • Z=Flow cytometric analysis of the sample on any flow cytometer capable of measuring scatter parameters and the labels employed.
  • Assembly 35: Identification of a group of entities using the same labeling molecules on different markers defining these entities.
  • A=Multiple marker molecules, all labeled with the same label molecule used for either positive or negative selection of entities.
  • Z=Flow cytometric analysis of the sample on any flow cytometer capable of detecting the label employed.
  • Assembly 36: Identification of a group of entities using the same labeling molecules on different markers defining these entities.
  • A=Multiple marker molecules, labeled with the same label molecule used for either positive or negative selection of entities of interest.
  • B=Multiple marker molecules, labeled with the same label molecule different from those used in “A”, used for either positive or negative selection of entities of interest.
  • Z=Flow cytometric analysis of the sample on any flow cytometer capable of detecting the label employed.
  • Assembly 37: Identification of a heterogeneous cell population in a sample.
  • A=Multiple marker molecules molecule specifically binding any of the specific cells in the cell population of interest.
  • B=A detection molecule specifically binding to any cell population different to the cell population of interest.
  • Z=Flow cytometric analysis of the sample on any flow cytometer capable of measuring scatter parameters and the labels employed.
  • Assembly 38: Identification of a cell population in a sample.
  • A=Multiple of marker molecules specifically binding any of the specific cells in the cell population of interest, labeled with the same label.
  • B=A number of marker molecules different from those used in “A” specifically binding any population of cells different from cells in “A”, labeled with the same label.
  • Z=Flow cytometric analysis of the sample on any flow cytometer capable of measuring the labels employed.
  • Assembly 39: Identification of specific CTL's (Cytotoxic T Lymphocytes) involved in the immune response against a specific protein or proteins.
  • A=Multiple MHC-dextramer reagents (more than one), each carrying a peptide homologues to a peptide sequence in the specific protein or proteins of interest, labeled with the same label.
  • Z=Flow cytometric analysis of the sample on any flow cytometer capable of measuring scatter parameters and the labels employed.
  • Assembly 40: Identification of specific CTL's (Cytotoxic T Lymphocytes) involved in the immune response against a specific protein or proteins.
  • A=Multiple MHC-dextramer reagents (more than one), each carrying a peptide homologues to a peptide sequence in the specific protein or proteins of interest, labeled with the same label.
  • B=A number of marker molecules different from those used in “A” specifically binding any population of cells different from cells defined by the markers used in “A”, labeled with the same label different form the label that labeling the MHC-dextramers.
  • C=Scatter parameters may be used in positive and or negative selection of cell populations.
  • Z=Flow cytometric analysis of the sample on any flow cytometer capable of measuring scatter parameters and the labels employed.
  • Assembly 41: Identification of specific CTL's (Cytotoxic T Lymphocytes) involved in the immune response against a specific protein or proteins.
  • A=Multiple MHC-dextramer reagents (more than one), each carrying a peptide homologues to a peptide sequence in the specific protein or proteins, of interest, labeled with the same label.
  • B=One or more than one labeled antibody, labeled with the same labeling molecule, different from that used for the MHC-dextramer, e.g. anti-CD8, and anti-CD3 markers.
  • C=Optional Scatter parameter
  • Z=Flow cytometric analysis of the sample on any flow cytometer capable of measuring scatter parameters and the labels employed.
  • Assembly 42: Identification of specific CTL's (Cytotoxic T Lymphocytes) involved in the immune response against a specific protein or proteins.
  • A=Multiple MHC-dextramer reagents (more than one), each carrying a peptide homologues to a peptide sequence in the specific protein or proteins, of interest, labeled with the same label.
  • B=CD8 antibody labeled with a labeling molecule different from that labeling the MHC-dextramer.
  • C=Optional a CD3 antibody labeled with a labeling molecule different form that used on the MHC-dextramer, and the CD8 antibody.
  • D=One or more than one labeled antibody, labeled with the same labeling molecule, different from that used for the MHC-dextramer, the anti-CD8, and the anti-CD3 markers, that are not recognizing the population of cells positive for the MHC reagent.
  • E=Optional Scatter parameter
  • Assembly 43: Identification of specific CTL's (Cytotoxic T Lymphocytes) involved in the immune response against a specific group of organisms, or antigens e.g. CMV, EBV, or BK family of viruses, or Tumor antigens.
  • A=Multiple MHC-dextramer reagents (more than one) labeled with the same label, each carrying a peptide homologues to a peptide sequence in proteins derived from group of organisms or tumor cells.
  • B=A marker molecule labeled with a label different form that used in A that are used to select of deselect entities in the sample analyzed.
  • C=Scatter parameters are optionally used in the selection process
  • Z=Flow cytometric analysis of the sample on any flow cytometer capable of measuring scatter parameters and the labels employed.
  • Assembly 44: Identification of specific CTL's (Cytotoxic T Lymphocytes) involved in the immune response against a specific group of organisms, or antigens e.g. CMV, EBV, or BK family of viruses, or Tumor antigens.
  • A=Multiple MHC-dextramer reagents (more than one) labeled with the same label, each carrying a peptide homologues to a peptide sequence in proteins derived from members of the family of organism or tumor cells.
  • B=CD8 antibody labeled with a labeling molecule different form that used on the MHC-dextramer.
  • C=One or more than one labeled antibodies identifying cell populations either different from the CTL's, all labeled with the same labeling molecule, different from that used for the MHC-dextramer, and the CD8 antibody.
  • D=Scatter parameter
  • Z=Flow cytometric analysis of the sample on any flow cytometer capable of measuring scatter parameters and the labels employed.
  • Assembly 45: Identification and activation analysis of specific CTL's (Cytotoxic T Lymphocytes) involved in the immune response against a pathogen family.
  • A=Multiple MHC-dextramer reagents (more than one) labeled with the same label, each carrying a peptide homologues to peptide sequences in proteins derived from members of the pathogen family.
  • B=CD8 antibody labeled with a labeling molecule different form that used on the MHC-dextramer.
  • C=One or more than one labeled antibodies indicative for activation status of CTL's labeled with a labeling molecule different from that used for the MHC-dextramer, and the CD8 antibody.
  • D=Scatter parameter, optionally
  • Z=Flow cytometric analysis of the sample on any flow cytometer capable of measuring scatter parameters and the labels employed.
  • Interpretation of Data and Manipulating Entities (E and F)
  • The output of the analysis and sorting (described in previous section) is analytical data and a population of entities, respectively. The data may be evaluated to obtain a final conclusion, e.g. a diagnosis for a patient. The cells may be further manipulated or analyzed; for example, the cells may be proliferated, matured and/or multiplied, for use in a therapeutic scheme for a patient.
  • Interpretation of Data to Diagnosis
  • In the case where the goal of the analysis is to obtain a diagnosis for a patient, after e.g. flow cytometry analysis of the patient's blood, the procedure may involve i) Examination of the acquired data by e.g. a physician, or software that performs an automated analysis of the data, to obtain a diagnosis for the patient. The data, the conclusion reached, and the general patient information may be stored or tightly linked, ii) The data may be stored centrally, in order to allow (secured) access from any computer on the www, enabling the data to be evaluated by individuals (e.g. a physician) not necessary present on the site where the data has been acquired, iii) The data and diagnosis may be linked to the “LIS” (laboratorie information system) system of the hospital, by specialized software.
  • In the case of an automated analysis, the sample may be phenotyped by cluster analysis of the data in n-dimensions, and compared to a reference, which typically will be a negative control as well as a statistical sample of healthy individuals. Evaluation of a statistical as well as a physical “healthy” sample enables the software to compare the value of the n parameters to the value typically obtained for healthy persons and patients, respectively, and/or compare to the cluster analysis of the healthy standard individual. Both approaches to data interpretaiton may form the basis of the diagnosis.
  • Manipulation of Selected Entities
  • The enriched population of entities that have been obtained from the sorting process, or by batch-based isolation may be further manipulated, as described by the following examples, which refer to the case where certain cells have been selected in the sorting process:
      • Cells may be expanded to a desired density
      • Cells may optionally be expanded, and assayed for cellular or other biochemical properties
      • Cells may optionally be expanded, and assayed for quantitative association with specific marker molecules.
      • Cells may be proliferated and/or matured by exposure to e.g. interleukins, growth factors, serum factors and/or hormones. The cells may then be used for re-administration into the patient from which the sample derives, or into other individuals, after a manipulation step.
      • Cells may optionally be expanded, and assayed in biochemical assays, such as phosphoylation status, enzymatic activity, and content of specific RNA, DNA or other cell components.
  • Examples of Optimized Methods for Detecting and Isolating Entities
  • Different analytical tasks require different approaches, and the individual approach must be optimized according to the desired outcome, both as regards the combination of sub-processes and as regards the optimization of the individual sub-processes.
  • All approaches, however, comprise one or more of the sub-processes (steps) Sampling (A)—Sample Preparation (B)—Assaying (C)—Data acquisition (D1)/Sorting (D2)—Interpretation of data (E)—Manipulating entities (F). As already indicated herein above, while steps A, B and C are essential, steps D (D1 and D2); E and F are optional method steps.
  • An illustration of one example of a “complete” method can thus be described e.g. as
  • An×Bn×Cn×Dn×En or Fn,
  • where n denote the totality of n different processes of a given category, including the choice of the sub-processes (embodiments of that particular method step) where the sub-process (embodiment) in question is not performed. n may be different for the individual sub-processes A, B, C, D, or E.
  • Below is Presented Examples of Some of the Different Sampling Processes Mentioned Above are:
  • A1: A sampling process where the source is blood
  • A2: A sampling process where the source is semen
  • A3: A sampling process where the source is human Blood
  • A4: A sampling process where the source is human cerebrospinal liquid
  • A5: A sampling process where the source is animal blood
  • A6: A sampling process where the source is animal semen
  • A7: A sampling process where the source is blood collected in a vacutainer®, a closed test tube, where the blood is drawn by a vacuum in the tube.
  • A8: A sampling process where the source is human blood collected in a vacutainer®, a closed test tube, where the blood is drawn by a vacuum in the tube.
  • A9: A sampling process where the source is animal blood collected in a vacutainer®, a closed test tube, where the blood is drawn by a vacuum in the tube.
  • A10: A sampling process where the source is blood collected in a capillary tube.
  • A11: A sampling process where the source is human blood collected in a capillary tube.
  • A12: A sampling process where the source is animal blood collected in a capillary tube
  • A13: A sampling process where the source is blood collected in a volumetric capillary tube, for a specific volume collection.
  • A14: A sampling process where the source is human blood collected in a volumetric capillary tube, for a specific volume collection.
  • A15: A sampling process where the source is animal blood collected in a volumetric capillary tube, for a specific volume collection.
  • A16: A sampling process where the source is human semen collected in a capillary tube.
  • A17: A sampling process where the source is animal semen collected in a capillary tube.
  • A18: A sampling process where the source is human semen collected in a volumetric capillary tube, for a specific volume collection.
  • A19: A sampling process where the source is animal semen collected in a volumetric capillary tube, for a specific volume collection.
  • A20: A sampling process where the source is blood collected in a vacutainer®, a closed test tube, where the blood is drawn by a vacuum in the tube, containing anticoagulating agent(s).
  • A21: A sampling process where the source is human blood collected in a vacutainer®, a closed test tube, where the blood is drawn by a vacuum in the tube, containing anticoagulating agent(s).
  • A22: A sampling process where the source is animal blood collected in a vacutainer®, a closed test tube, where the blood is drawn by a vacuum in the tube, containing anticoagulating agent(s).
  • A23: A sampling process where the source is blood collected in a capillary tube, containing anticoagulating agent(s).
  • A24: A sampling process where the source is human blood collected in a capillary tube, containing anticoagulating agent(s).
  • A25: A sampling process where the source is animal blood collected in a capillary tube, containing anticoagulating agent(s).
  • A26: A sampling process where the source is blood collected in a volumetric capillary tube, for a specific volume collection, containing anticoagulating agent(s).
  • A27: A sampling process where the source is human blood collected in a volumetric capillary tube, for a specific volume collection, containing anticoagulating agent(s).
  • A28: A sampling process where the source is animal blood collected in a volumetric capillary tube, for a specific volume collection, containing anticoagulating agent(s).
  • A29: A sampling process where the source is human semen collected in a capillary tube.
  • A30: A sampling process where the source is animal semen collected in a capillary tube.
  • A31: A sampling process where the source is human semen collected in a volumetric capillary tube, for a specific volume collection.
  • A32: A sampling process where the source is animal semen collected in a volumetric capillary tube, for a specific volume collection.
  • A33: A sampling process where the source is liquefied solid tissue suspension
  • A34: A sampling process where the source is human liquefied solid tissue suspension
  • A35: A sampling process where the source is animal liquefied solid tissue suspension
  • A36: A sampling process where the source is cultures of bacteria
  • A37: A sampling process where the source is cultures of yeast
  • A38: A sampling process where the source is fermenting cultures of bacteria, to follow the growth and composition of the culture.
  • A39: A sampling process where the source is fermenting cultures of yeast, to follow the growth and composition of the culture.
  • A40: A sampling process where the source is chemical reactions mixtures
  • A41: A sampling process where the source is a chemical reactions mixtures, collected in a tube or capillary tube containing particles that enables assaying a component in the reaction mixture.
  • A41: A sampling process where the source is a chemical reactions mixtures, collected in a tube or capillary tube containing chemical reagent or reagents that enables assaying a component, or components in the reaction mixture.
  • A42: A sampling process where the source is drinking water.
  • A43: A sampling process where the source is drinking water collected in a tube containing reagents for a specific assay of the water content.
  • A44: A sampling process where the source is sewage.
  • A45: A sampling process where the source is sewage collected in a tube containing reagents for a specific assay of the content.
  • A46: A sampling process where the source is soil.
  • A47: A sampling process where the source is soil collected and resuspended, in a tube containing a specific reagent or reagents that enables assaying a component, or components in the soil sample.
  • A48: A sampling process where the source is urine, collected in a tube or capillary tube
  • A49: A sampling process where the source is urine, collected in a tube or capillary tube, containing a specific reagent or reagents that enables assaying a component, or components in the sample.
  • A50: A sampling process where the source is urine samples in a volumetric tube or capillary tube, containing a specific reagent or reagents that enables assaying a component, or components in the sample.
  • A51: A sampling process where the source is human urine collected in a volumetric tube or capillary tube, containing a specific reagent or reagents that enable assaying a component, or components in the sample.
  • A52: A sampling process where the source is urine, collected in a tube or capillary tube, for analysis using a flowcytometry based instrument
  • A53: A sampling process where the source is human urine collected in a volumetric tube or capillary tube, containing a specific reagent or reagents that enables assaying a component, or components in the sample, for analysis using a flowcytometry based instrument.
  • A54: A sampling process where the source is blood collected in a vacutainer®, a closed test tube, where the blood is drawn by a vacuum in the tube, containing anticoagulating agent, as EDTA, or Citrate, and antioxidants.
  • A55: A sampling process where the source is human blood collected in a vacutainer®, a closed test tube, where the blood is drawn by a vacuum in the tube, containing anticoagulating agent, as EDTA, or Citrate, and antioxidants.
  • A56: A sampling process where the source is animal blood collected in a vacutainer®, a closed test tube, where the blood is drawn by a vacuum in the tube, containing anticoagulating agent, as EDTA, or Citrate, and antioxidants.
  • A57: A sampling process where the source is blood collected in a capillary tube, containing anticoagulating agent, as EDTA, Citrate or Heparins, and antioxidants.
  • A58: A sampling process where the source is human blood collected in a capillary tube, containing anticoagulating agent, as EDTA, Citrate or Heparins, and antioxidants.
  • A59: A sampling process where the source is animal blood collected in a capillary tube, containing anticoagulating agent, as EDTA, Citrate or Heparins, and antioxidants.
  • A60: A sampling process where the source is human semen collected in a tube, containing antifreeze—agents as DMSO or glyserol.
  • A61: A sampling process where the source is animal semen collected in a tube, containing antifreeze—agents as DMSO or glyserol.
  • A62: A sampling process where the source is human semen collected in a capillary tube, containing “antifreeze-agents” as DMSO or glycerol.
  • A63: A sampling process where the source is animal semen collected in a capillary tube, containing “antifreeze-agents” as DMSO or glycerol.
  • A64: A sampling process where the source is blood collected in a donor blood-bag, containing anticoagulating agent, as EDTA, Citrate or Heparins, and antioxidants.
  • A65: A sampling process where the source is blood collected in a disposal container, containing anticoagulating agent, as EDTA, Citrate or Heparins, and antioxidants.
  • A66: A sampling process where the source is blood collected in container, containing fixative.
  • A67: A sampling process where the sample is collected in a volumetric container, containing internal standards, such as counting beads.
  • A68: A sampling process where the sample is collected in a volumetric container, containing reagents for the further analysis.
  • A69: A sampling process where the sample is collected in a container that is coated with a marker molecule selectively binding a specific component or components of the sample, enriching for the unbound entities in the suspension.
  • A70: A sampling process where the source is a sample collected in a container that is labeled with a RIF based chip or otherwise labeled, enabling tracking of the source during the different step in the analysis procedure, as well as the treatment that sample has gone through.
  • A71:A sampling process where the source is blood collected in a vacutainer®, a closed test tube, where the blood is drawn by a vacuum in the tube, containing anticoagulating agent, as EDTA, or Citrate, and also cell separation medium e.g. comprised of a polyester gel and a density gradient liquid.
  • A72: A sampling process where the source is human blood collected in a vacutainer®, a closed test tube, where the blood is drawn by a vacuum in the tube, containing anticoagulating agent, as EDTA, or Citrate, and antioxidants and also cell separation medium e.g. comprised of a polyester gel and a density gradient liquid
  • A73: A sampling process where the source is animal blood collected in a vacutainer®, a closed test tube, where the blood is drawn by a vacuum in the tube, containing anticoagulating agent, as EDTA, or Citrate, and also cell separation medium e.g. comprised of a polyester gel and a density gradient liquid.
  • A74: A sampling process where the source is blood collected in a capillary tube, containing anticoagulating agent, as EDTA, Citrate or Heparins, and also cell separation medium e.g. comprised of a polyester gel and a density gradient liquid.
  • A75: A sampling process where the source is human blood collected in a capillary tube, containing anticoagulating agent, as EDTA, Citrate or Heparins, and also cell separation medium e.g. comprised of a polyester gel and a density gradient liquid.
  • A76: A sampling process where the source is animal blood collected in a capillary tube, containing anticoagulating agent, as EDTA, Citrate or Heparins, and also cell separation medium e.g. comprised of a polyester gel and a density gradient liquid.
  • A77:A sampling process where the source is blood collected in a vacutainer®, a closed test tube, where the blood is drawn by a vacuum in the tube, containing anticoagulating agent, as EDTA, or Citrate, and antioxidants, and also cell separation medium e.g. comprised of a polyester gel and a density gradient liquid.
  • A78: A sampling process where the source is human blood collected in a vacutainer®, a closed test tube, where the blood is drawn by a vacuum in the tube, containing anticoagulating agent, as EDTA, or Citrate, and antioxidants, and antioxidants and also cell separation medium e.g. comprised of a polyester gel and a density gradient liquid
  • A79: A sampling process where the source is animal blood collected in a vacutainer®, a closed test tube, where the blood is drawn by a vacuum in the tube, containing anticoagulating agent, as EDTA, or Citrate, and antioxidants, and also cell separation medium e.g. comprised of a polyester gel and a density gradient liquid.
  • A80: A sampling process where the source is blood collected in a capillary tube, containing anticoagulating agent, as EDTA, Citrate or Heparins, and antioxidants, and also cell separation medium e.g. comprised of a polyester gel and a density gradient liquid.
  • A81: A sampling process where the source is human blood collected in a capillary tube, containing anticoagulating agent, as EDTA, Citrate or Heparins, and also cell separation medium e.g. comprised of a polyester gel and a density gradient liquid.
  • A82: A sampling process where the source is animal blood collected in a capillary tube, containing anticoagulating agent, as EDTA, Citrate or Heparins, and antioxidants, and also cell separation medium e.g. comprised of a polyester gel and a density gradient liquid.
  • Below is Presented Examples of Some of the Different Sample Preparation Procedures Mntioned Above is:
  • B1: A sample preparation where the sample is added reagents that protect the sample against oxidation.
  • B2: A sample preparation where the sample is added reagents that stabilize the sample, and protect against cell lysis, such as fixatives and sugar.
  • B3: A sample preparation where the sample is added biocides or fixation to protect the operator form potentially contamination danger, such as viral or bacterial infections.
  • B4: A sample preparation where the sample is treated for selectively enrichement of a sub component of the sample.
  • B5: A sample preparation where the sample is treated with a chemical that lyse a specific subpopulation of cells in the sample, such as lysis of RBC in full blood using UtiLyse™, EasyLyse™, FACSLyse™, or other ammonium containing solutions and/or by osmotic shock.
  • B6: A sample preparation where the sample is treated with a chemical that precipitate a specific sup-population of the cells, such as antibody mediated aggregation, or association with beads having a specific marker molecule on the surface.
  • B7: A sample preparation where the sample is fractionated by centrifugation in a density mediating substance, such as ficoll, sucrose or glycerol, separating different entity- populations by their density.
  • B8: A sample preparation where the sample is fractionated by centrifugation through a mesh membrane separating smaller from larger entities.
  • B9: A sample preparation where the sample is exposed to magnetized beads linked to a marker molecule that specifically bind a specific entity, selective retained in a magnetic field.
  • B10: A sample preparation where the sample is added fluorescent beads for internal standards during the analysis of the sample.
  • B11: A sample preparation where a specific volume of the sample is added a specific number of beads for quantitative counting of entities in the sample.
  • B12: A sample preparation where the sample is added specific marker molecules, such as antibodies for detection of specific entities within the sample.
  • B13: A sample preparation where the sample is added specific detections molecules allowing for phenotyping of the sample entities, by detecting the labelling on the detection molecules in an appropriate assay.
  • B14: A sample preparation where the sample is filtered to remove debris that potentially could interfere with the analysis method, such as flowcytometry where debris can block the sample probe.
  • B15: A sample preparation where the sample is added into a microtitreplate, or test tube containing immobilized detection molecules such as labeled antibodies in a matrix of sugars.
  • B16: A sample preparation where the sample is added reagents by an automated pippetting device and subsequently automatically applied to the flowcytometer or another analysis instrument.
  • B17: A sample preparation where the sample is added a particle capeable of association with the detections molecules as a procedure control.
  • B18: A sample preparation where solid tissue is mechanical and enzymatical processed to produce a cell suspension, a sample applicable to flowcytometry after incubation with specific detection molecules.
  • B19: A sample preparation where the source is solid parafin fixed tissue, sliced, placed on coverglass, and used as sample, prepared for assaying by de-parafination, antigen retrieval and subsequent incubated with detection molecules.
  • B20: A sample preparation where the source is frozen tissue, sliced, placed on a coverglass, fixed using alcohol and used as sample, assayed by incubated with detection molecules.
  • B21: A sample preparation where the source is a cell suspension, smeared onto a coverglass, fixed using alcohol and used as sample and assayed by incubated with detection molecules. Some of the Assay Procedures Mentioned Above is:
  • C1: Analysis of the prepared sample using Cytometry, as assay procedure.
  • C2: Analysis of the prepared sample using Cytometry as assay procedure where the detection is based on the intensity of the fluorochromes on the marker molecules.
  • C3: Analysis of the prepared sample using Cytometry as assay procedure where the detection is based on the halflife, (fluorescence decay time), of the fluorochromes on the marker molecules.
  • C4: Analysis of the prepared sample using Cytometry as assay procedure where the detection is based on the total spectra of the fluorochrome, measured by multi-channel PMT or photo-responsive chip technology.
  • C5: Analysis of the prepared sample using Stationary Cytometry
  • C6: Analysis of the prepared sample using Stationary Cytometry, where detection is based on the enzymatic generation of precipitated dye molecules, or light emission.
  • C7: Analysis of the prepared sample using Stationary Cytometry, where detection is based on fluorescence labeled marker molecules, detected using a digitalized picture of the labeled sample.
  • C8: Analysis of the prepared sample using sheath fluid based flowcytometry as assay procedure.
  • C9: Analysis of the prepared sample using “dry” (e.g.flow-on-a-chip) flowcytometry, as assay procedure.
  • C10: Analysis of the prepared sample using capillary based flowcytometry, as assay procedure.
  • C10: Analysis of the prepared sample using immunohistochemestry (IHC), as assay procedure.
  • C12: Analysis of the prepared sample using batch-based assay
  • C13: Analysis of the prepared sample using batch-based assay, where a specific marker molecules are immobilized on a solid support and used to capture the specific population of entities associating with it.
  • C14: Analysis of the prepared sample using batch-based assay, where a specific marker molecules are immobilized on a particle and used to capture the specific population of entities associating with it, subsequent detected be a second detections molecule using flowcytometry as final assay.
  • C15: Analysis of the prepared sample using batch-based assay, where a specific marker molecules are immobilized on defined arrears of a surface and used to capture the specific population or populations of entities associating with them, detected with a generic cell dye.
  • C16: Analysis of the prepared sample using batch-based assay, where a specific marker molecules are immobilized on defined arrears of a surface and used to capture the specific population or populations of entities associating with these arrears, detected with a specific detection molecule.
  • C17: Analysis of the prepared sample using batch-based assay, where a specific marker molecules are immobilized on a solid support and used to capture the population of entities associating with it, detected using an intrinsic property of the cells, such as an enzymatic activity, or a colour reaction with a cellular component.
  • Some of the Assay Procedures Used in Sorting, Mentioned Above is:
  • C18: Sorting of the prepared sample using Cytometry.
  • C19: Sorting of the prepared sample using Cytometry, where the detection is based on the intensity of the fluorochromes on the detection molecules.
  • C20: Sorting of the prepared sample using Cytometry, where the detection is based on the half-life (fluorescence decay time), of the fluorochromes on the marker molecules.
  • C21: Sorting of the prepared sample using Cytometry, where the detection is based on the total spectra of the fluorochrome, measured by multi-channel PMT or photo-responsive chip technology.
  • C22: Sorting of the prepared sample using sheath fluid based flowcytometry.
  • C23: Sorting of the prepared sample using “dry” (e.g. flow-on-a-chip) flowcytometry.
  • C24: Sorting of the prepared sample using capillary based flowcytometry.
  • C25: Sorting of the prepared sample using batch-based assay
  • C26: Sorting of the prepared sample using batch-based assay, where a specific marker molecules are immobilized on a solid support and used to capture the specific population of entities associating with it.
  • C27: Sorting of the prepared sample using batch-based assay, where a specific marker molecules are immobilized on a particle and used to capture the specific population of entities associating with it by centrifugation.
  • C28: Sorting of the prepared sample using batch-based assay, where a specific marker molecules are immobilized on magnetic particle used to capture the specific population of entities associating it, by means of a magnetic field.
  • Some of the Data Analysis Procedures Described Above are:
  • D1: Data generated using flowcytometry are analyzed for the presence of MHC-mer mediated fluorescing entities in the sample.
  • D2: The data generated using flowcytometry, are analyzed for the presence of MHC-mer mediated and anti-CD8 mediated double fluorescing entities in the sample.
  • D3: The data generated using flowcytometry, are analyzed for the presence of MHC-mer mediated, anti-CD8 mediated and anti-CD3 mediated triple fluorescing entities in the sample.
  • D4: The data generated using flowcytometry, are analyzed for the presence of MHC-mer mediated, anti-CD8 mediated and anti-CD3 mediated triple fluorescing entities in the sample that are identified as a subpopulation of the anti-CD45 mediated fluorescing population.
  • D5: The data generated using flowcytometry are analyzed using a “dump” gate strategy in which the MHC-mer mediated fluorescing entities are defined by not being labeled with certain makers that are known to define population of entities in which the MHC-mer binding entities will not be found.
  • D6: The data generated using flowcytometry are analyzed for the presence of MHC-mer mediated fluorescing entities in the sample, among entities defined by not being labeled with CD4 fluorescing antibodies.
  • D7: The data generated using flowcytometry are analyzed for the presence of MHC-mer mediated fluorescing entities in the sample, among the entities defined by not being labeled with CD4, CD19, or CD15 fluorescing antibodies.
  • D8: The data generated using flowcytometry are analyzed for the presence of MHC-mer mediated fluorescing entities in the sample, among the entities defined by not being labeled with all or a subset of CD4, CD14, CD15 CD19, and CD71 fluorescing antibodies and staining that defined the dead cells in the sample.
  • D9: The data generated using flowcytometry are analyzed for the presence entities defined by binding to MHC-mer detected by FITC fluorescence.
  • D10: The data generated using flowcytometry are analyzed for the presence entities defined by binding to MHC-mer detected by RPE fluorescence.
  • D11: The data generated using flowcytometry are analyzed for the presence entities defined by binding to MHC-mer detected by APC fluorescence.
  • D12: The data generated using flowcytometry are analyzed for the presence entities defined by binding to MHC-mer detected by PB (pacific blue) fluorescence.
  • D13: The data generated using flowcytometry are analyzed for the presence of entities defined by binding to MHC-mer detected by e.g labels exmplified in Table 3a and b.
  • D14: The data generated using flowcytometry are analyzed for the concentration of entities defined by binding to MHC-mer detected by e.g labels exmplified in Table 3a and b relative to the amount of a counting bead such as CytoCount™.
  • D15: The data generated using flowcytometry are analyzed for the present of a solid phase fluorescent bead or beads each associated with a specific MHC-mer molecule, which has captured an antigen specific T-cell detected by a fluorescent generic cell stain.
  • D16: The data generated using flowcytometry are analyzed for the present of a solid phase fluorescent bead or beads each associated with a specific MHC-mer molecule, which has captured an antigen specific T-cell detected by a fluorescent DNA stain, such as Draq5 or Propidium iodide.
  • D17: The data generated using flowcytometry are analyzed for the present of a solid phase fluorescent bead or beads each associated with a specific MHC-mer molecule, which has captured an antigen specific T-cell detected by a fluorescent detection molecule staining other characteristic of the specific T cell population, such as CD8.
  • D18: Identification of antigen specific T-cells by a solid phase surface assay, for the presence of cells in an area coated with a specific MHC-mer molecule, by staining the cells with a generic cell stain such a protein stain.
  • D19: Identification of antigen specific T-cells by a solid phase surface assay, for the presence of cells in an area coated with a specific MHC-mer molecules, by staining of bound cells using a fluorescent detection molecule detecting other characteristic of the specific T cell population, such as CD8.
  • Some of the Procedures for Handling the Products or Isolated Cells are:
  • E1: Based on the analysed data from flowcytometry, the amount of a specific entity is calculated relative to another defined entity in the sample.
  • E2: Based on the analysed data from flowcytometry, the amount of an antigen specific T-cell is calculated relative to another defined cell type in the sample.
  • E3: Based on the analysed data from flowcytometry, the amount of an antigen specific T-cells are calculated relative to the amount of CD8 positive cells in the sample.
  • E4: Based on the analysed data from volumetric flowcytometry, the amount of a specific entity is calculated relative to the volume of sample analysed, giving a concentration of the specific cell/μl sample.
  • E5: Based on the analysed data from volumetric flowcytometry, the amount of an antigen specific T-cells is calculated relative to the volume of sample analysed, giving a concentration of the specific cell/μl sample.
  • E6: Based on the analysed data from flowcytometry, the amount of a specific entity is calculated relative to a known amount of beads added to the sample, such as CytoCount™, giving the concentration of the specific entity in the sample, also called “one platform counting”.
  • E7: Based on the analysed data from flowcytometry, the amount of an antigen specific T-cell is calculated relative to a known amount of beads added to the sample, such as CytoCount™, giving the concentration of this antigen specific T-cell in the sample.
  • E8: Based on the analysed data from flowcytometry, the amount of an antigen specific T-cell is calculated relative to a known amount of a constant cell population in the sample, such as the blood plates, giving the concentration of this antigen specific T-cell in the sample.
  • E9: The sorted cells are expanded in-vitro, and used in further characterization studies.
  • E10: The sorted antigen specific T-cells are expanded in-vitro, and re-introduced into the patients blood stream for increased immunity towards the specific antigen.
  • E11: The sorted antigen specific T-cells are expanded in-vitro, and re-introduced into the patients blood stream to be used as autologous cancer therapy.
  • 1: Specific T-cell analysis in human peripheral blood.
  • A (sampling): Use of vacutainer to collect 5 mL human blood.
  • B (sample preparation): 100 μl of full blood is transferred to a tube containing MHC HLA-A*0201/APC loaded with the peptide “NLVPMVATV” from the CMV virus and incubated for 10′ at room temperature in the dark, where after the gating reagents anti-CD3/FITC, anti-CD8/PB are Presently preferred methods include but is not limited to the following:
  • Process added followed by additional incubation and the subsequent lysis of the RBC using the reagent EasyLyse, followed by a centrifugation and resuspension of the cells in buffer, thus a Lyse wash procedure. For compensation controls the same procedure are performed with a anti-CD3/fluor staining for each fluor used in the experiment. C (assaying): The prepared sample is applied to an CyAn ADP™ flow cytometer, and the data acquired in an FCS file.
  • D (data analysis): The obtained flow cytometry data are analysed, first the spectral overlap are compensated for, using the Summit™ auto comp feature on the data from anti-CD3/fluor single stained samples.
  • The compensated data are analysed for the presence of cells that are triple stained with anti-CD3, anti-CD8 and MHC-mer reagents.
  • E (interpretation of data): The number of anti-CD8 (and anti-CD3) stained cells and that of anti-CD8, MHC-mer (and anti-CD3) stained cells are determined. The frequency of specific MHC-mer binding cells, or antigen specific T-cells towards the CMV virus specific peptide is calculated as “ # of MHC-mer cells/total # of CD8 positive cells”.
  • Process 2: Concentration of Specific T-Cell in Human Peripheral Blood.
  • A (sampling): Use of vacutainer to collect 5 mL human blood.
  • B (sample preparation): 100 μl of full blood is transferred to a tube containing MHC HLA-A*0201/RPE, loaded with the peptide “NLVPMVATV” from the CMV virus, and exactly 100 μl CytoCount™ bead suspension, subsequently the sample is incubated for 10′ at room temperature in the dark, where after the gating reagents anti-CD3/FITC, anti-CD8/APC are added followed by additional incubation and subsequent lysis of the RBC using the reagent EasyLyse™, thus a lyse no wash procedure. For compensation controls the same procedure are performed with a anti-CD3/fluor staining for each fluor used in the experiment, without the counting beads.
  • C (Assaying): The prepared sample is applied to an CyAn ADP™ flow cytometer, and the data acquired in an FCS file.
  • D (data analysis): The obtained flow cytometry data are analysed, first the spectral overlap are compensated for, using the Summit™ auto comp sorftware, on data from anti-CD3/fluor single stained samples.
  • The compensated data from the MHC-mer stainings are analysed for the presence and number of cells that are triple stained with anti-CD3, anti-CD8 and MHC-mer reagents, and that of the CytoCount beads acquired.
  • E (interpretation of data): The number of anti-CD3, anti-CD8, and MHC-mer triple stained cells is determined, as well as the number of CytoCount bead. The concentration of the specific MHC-mer binding cells, or antigen specific T-cells towards the CMV virus specific peptide in the full blood sample is calculated;
  • Cspecific T-cell=(Counted #SpecificTcell×Concbead stock)/Counted #beads acquired, where the concentration of beads are app. 1000 beads/μl stated on the vial, the volume of the CytoCount™ beads and the blood in the sample is the same.
  • Process 3: Concentration of Specific T-Cell in Human Peripheral Blood in a No Lyse Assay.
  • A (sampling): Use of vacutainer to collect 5 mL human blood, transfer 100 μl of the blood to “matrix prepared reagents multititter plate”, containing the solidified reagents.
  • B (sample preparation): The matrix prepared reagents wells or tubes contain the reagents, one tube contains MHC HLA-A*0201/APC, loaded with the peptide “NLVPMVATV” from the CMV virus, and the gating reagents anti-CD3/RPE, anti-CD8/Alexa700, and anti-CD45/PB, as well as the “dump” gate reagents anti-CD4, anti-CD14, anti-CD15, anti- anti-CD19, and anti-CD71 all labeled with FITC, and 1,00×105 CytoCount™ beads.
  • Some individual wells contain the compensation control reagents, anti-CD3/fluor staining for each fluor used in the experiment, without the counting beads.
  • After addition of blood, the samples are incubated for 15′ in the dark.
  • C (Assaying): The prepared samples are applied to a CyAn ADP™ flow cytometer, and the data acquired in an FCS file.
  • D (data analysis): The obtained flow cytometry data are analysed; first the spectral overlap are compensated for, using the SummitlM auto comp sorftware, on data from anti-CD3/fluor single stained samples.
  • The cells that are stained for FITC (the dump gate reagents), are gated away and the anti-CD45/PB positive cells are selected for further analysis and gating.
  • The anti-CD45/PB positive cells from the MHC-mer stainings are analysed for the presence and number of cells that are triple stained with anti-CD3, anti-CD8 and MHC-mer reagents.
  • The amount of CytoCount beads acquired is likewise determined for each sample.
  • E (interpretation of data): The number of anti-CD45, anti-CD3, anti-CD8, and MHC-mer triple stained cells are determined, as well as the number of CytoCount bead. The concentration of the specific MHC-mer binding cells, or antigen specific T-cells towards the CMV virus specific peptide in the full blood is calculated;
  • Cspecific T-cell=Dilution factor of blood×(Counted #specific T cell×Concbead stock)/Counted #beads acquired, where the concentration of beads are app. 1000 beads/μl stated on the vial, and the dilutions factor in this experiment setup is 2.
  • Process 4: Isolation of Specific T-Cell in Human Peripheral Blood
  • A (sampling): Use of vacutainer to collect 5 mL human blood.
  • B (sample preparation): 2.5 ml of full blood is transferred to a tube containing MHC-mer/APC loaded with a specific peptide from the protein of interest and incubated for 10 min at room temperature in the dark, where after the gating reagents anti-CD3/FITC, anti-CD8/PB are added followed by additional incubation and the subsequent lysis of the RBC using the reagent EasyLyse, followed by a centrifugation and resuspension of the cells, thus a Lyse wash procedure. For compensation controls 100 μl blood are stained individually with anti-CD3/fluor for each fluor used in the experiment.
  • C (assaying): The prepared sample is applied to an MoFLo™ cell sorter and the data from the sort are stored in an FCS file.
  • D (Sorting): The preliminary flow cytometry data are analysed, first the spectral overlap are compensated for, using the Summit™ auto comp sorftware on the data from anti-CD3/fluor single stained samples.
  • The compensated data are analysed for the presence of cells that are triple stained with anti-CD3, anti-CD8 and MHC-mer reagents, and the sort gate are placed accordingly.
  • The cells that are falling within the sortgate will be sorted into a cell tube containing growth medium and serum.
  • E (entity enrichment): The sorted cells are directly assayed again using a CyAn™ ADP™ flow cytometer, and the purity of the sample are judghed. If the purity is not sufficient for the purpose of the cells, a additional round of sorting can be performed, identical to the first.
  • When purity is satisfactory, the sorted antigen specific T-cells can be enriched by growth in-vitro, and subsequent administrated to the patient potentially increasing the immunity towards the specific protein represented by the peptide.
  • Process 5: Isolation of Antigen Specific T-Cells by Magnetic Beads
  • A (sampling): 500 ml blood is sampled in a blood donor bag
  • B (sample preparation): Magnetic particles are coupled or associated with MHC multimer loaded with a specific peptide from a protein of interest. The MHC multimer carrying magnetic particles are added into the donor blood bag, and incubated while mixing.
  • C/D (assaying and sorting): The blood bag is placed in a magnetic field, withholding the magnetic beads and the cells associating with them in the bag while the remainig blood are drained out.
  • The magnetic beads and associated cells is washed, and by removing the magnetic field, the beads and associated cells are freed and can be resuspended into a growth medium, or used for other purposes.
  • The cells associated with the beads can be tested in conventional flow Cytometry, as described in the former processes for identification control of the harvested cells.
  • E (entity enrichment): The enriched antigen specific T-cells can be expanded by growth in-vitro, and subsequent administrated to the patient potentially increasing the immunity towards the specific protein represented by the peptide.
  • Process 6: Detection of Antigen Specific T-Cells in Solid Tissue
  • A (sampling): Tissue, fixed and embedded in paraffin are sliced in thin slices and placed on a glass slice.
  • B (sample preparation): The paraffin block is sliced and thin slices are plased on glass slices. The sample is deparafinated, follow by a antigen retrival, and stained for DNA using DAPI and subsequent incubation with detection reagents, substrate is added and allowed to react.
  • C (assaying): The markers used are; a antigen specific MHCmer/HRP reagent (Horse Radish Peroxidase coubled), and DAPI stain for cell identification. The presence of antigen specific T cells is detected by the presipitation of a dye due to the HRP enz.
  • D (Data acquistion): The sample is analyzed manually by light microscopy, or by digital imaging.
  • E (Interpretation of Data): The amount of antigen specific T cells are determined by the precipitated color, correlated to the DAPI stain for positive cell identification, either manually of by automated software working on the digital image.
  • Process 7: Detection of Borrelia Specific T Cells in Human Blood.
  • A (sampling): Use of vacutainer or other container containing anti-coagulant to collect 4-10 ml human blood.
  • B (sample preparation): 100 μl of the full blood is transferred to a tube containing one or more MHC dextramers labeled with APC and carrying antigenic peptides derived from proteins from Borrelia bacteria. MHC dextramers are MHC-peptide complexes e.g. generated by in vitro refolding, coupled to dextran through covalent or non-covalent interactions. The dextramer furthermore comprises APC coupled to the same dextran molecules through covalent or non-covalent interactions. The MHC dextramers may all carry the same MHC I allele or carry different MHC I alleles.
  • Borrelia bacteria here include any strain of the species Borrelia Burgdorferi, Borrelia Garinii or Borrelia Afzelii. Example Borrelia proteins include but is not limited to OspA, OspB, OspC, OspE, OspG, VIsE, FlaB, FIaA, BmpA, BmpB, BmpC, BmpD, HSP90, CRASP-1, CRASP-2, DbpA, DbpB, MaIQ, BapA, other outer surface Borrelia proteins, other intracellular proteins of Borrelia and plasmid encoded proteins from Borrelia bacteria.
  • The antigenic peptides are preferably 9′mers but can also be 8′mers, 10′ers or 11′mers.
  • The MHC dextramers are incubated for 10′ at room temperature in the dark, then gating reagents including anti-CD3/PE, anti-CD8/PB and anti-CD4/FITC are added and incubation continued for 20′. RBC is lysed by addition of lysis reagents e.g. EasyLyse then the sample is centrifuged, supernatant removed. Cells are washed by resuspension in buffer followed by centrifugation. Supernatant is removed and cells resuspended in buffer.
  • Optionally fixative is added.
  • C (assaying): The prepared sample is applied to a flow cytometer and data acquired.
  • D (data analysis): The obtained data are analyzed for the presences of cells stained with MHC dextramer, anti-CD3, anti-CD8 and not stained with anti-CD4.
  • E (interpretation of data): The presence of cells stained with MHC dextramer, anti-CD3, anti-CD8 and not stained with anti-CD4 are T cells specific for Borrelia antigens and their frequency can optionally be determined as follows. The number of anti-CD8 and anti-CD3 stained cells and that of anti-CD8, MHC dextramer and anti-CD3 stained cells are determined. The frequency of specific MHC dextramer binding cells, or antigen specific T-cells towards Borrelia specific peptide is calculated as “ # of MHC dextramer positive, CD3+, CD8+cells/total # of CD3+,CD8 positive cells”.
  • Process 8: Detection of Borrelia Specific T Cells in Human Blood Using MHC Dextramers
  • A (sampling): Use of vacutainer or other container containing anti-coagulant to collect 4-10 ml human blood.
  • B (sample preparation): 10-500 μl of the full blood is transferred to a tube containing one or more MHC dextramers labeled with fluorochrome and carrying antigenic peptides derived from proteins from Borrelia bacteria. MHC dextramers are MHC-peptide complexes e.g. generated by in vitro refolding, coupled to dextran through covalent or non-covalent interactions. The dextramer furthermore comprises fluorochrome coupled to the same dextran molecules through covalent or non-covalent interactions. The MHC dextramers may all carry the same MHC I allele or carry different MHC I alleles.
  • Borrelia bacteria here include any strain of the species Borrelia Burgdorferi, Borrelia Garinii or Borrelia Afzelii. Example Borrelia proteins include but is not limited to OspA, OspB, OspC, OspE, OspG, VIsE, FlaB, FIaA, BmpA, BmpB, BmpC, BmpD, HSP90, GRASP-1, CRASP-2, DbpA, DbpB, MaIQ, BapA, other outer surface Borrelia proteins, other intracellular proteins of Borrelia and plasmid encoded proteins from Borrelia bacteria.
  • The antigenic peptides are preferably 9′mers but can also be 8′mers, 10′ers or 11′mers.
  • The MHC dextramers are incubated for 1-60′ at room temperature in the dark, then gating reagents including labeled markers directed against CD3, CD8 and CD4 are added and incubation continued for 1-60′. RBC is lysed by addition of lysis reagents e.g. EasyLyse then the sample is centrifuged, supernatant removed.
  • Cells are washed by resuspension in buffer followed by centrifugation. Supernatant is removed and cells resuspended in buffer.
  • Optionally fixative is added.
  • C (assaying): The prepared sample is applied to a flow cytometer and data acquired.
  • D (data analysis): The obtained data are analyzed for the presences of cells stained with MHC dextramer, anti-CD3, anti-CD8 and not stained with anti-CD4.
  • E (interpretation of data): The presence of cells stained with MHC dextramer, anti-CD3, anti-CD8 and not stained with anti-CD4 are T cells specific for Borrelia antigens and their frequency can optionally be determined as follows. The number of anti-CD8 and anti-CD3 stained cells and that of anti-CD8, MHC dextramer and anti-CD3 stained cells are determined. The frequency of specific MHC dextramer binding cells, or antigen specific T-cells towards Borrelia specific peptide is calculated as “# of MHC dextramer positive, CD3+, CD8+cells/total # of CD3+, CD8 positive cells”.
  • Process 9: Detection of Borrelia Specific T Cells in Human Blood Using a Lyse No-Wash Method.
  • A (sampling): Use of vacutainer or other container containing anti-coagulant to collect 4-10 ml human blood.
  • B (sample preparation): 10-500 μl of the full blood is transferred to a tube containing one or more MHC dextramers labeled with fluorochrome and carrying antigenic peptides derived from proteins from Borrelia bacteria. MHC dextramers are MHC-peptide complexes e.g. generated by in vitro refolding, coupled to dextran through covalent or non-covalent interactions. The dextramer furthermore comprises fluorochrome coupled to the same dextran molecules through covalent or non-covalent interactions. The MHC dextramers may all carry the same MHC I allele or carry different MHC I alleles.
  • Borrelia bacteria here include any strain of the species Borrelia Burgdorferi, Borrelia Garinii or Borrelia Afzelii. Example Borrelia proteins include but is not limited to OspA, OspB, OspC, OspE, OspG, VIsE, FlaB, FIaA, BmpA, BmpB, BmpC, BmpD, HSP90, GRASP-1, CRASP-2, DbpA, DbpB, MaIQ, BapA, other outer surface Borrelia proteins, other intracellular proteins of Borrelia and plasmid encoded proteins from Borrelia bacteria.
  • The antigenic peptides are preferably 9′mers but can also be 8′mers, 10′ers or 11′mers.
  • The MHC dextramers are incubated for 1-30′ at room temperature in the dark, then gating reagents including labeled markers directed against CD3, CD8 and CD4 are added and incubation continued for 1-60′. RBC is lysed by addition of lysis reagents e.g. EasyLyse and buffer is added to dilute sample 2-50 times.
  • Optionally fixative is added.
  • C (assaying): The prepared sample is applied to a flow cytometer and data acquired.
  • D (data analysis): The obtained data are analyzed for the presences of cells stained with MHC dextramer, anti-CD3, anti-CD8 and not stained with anti-CD4.
  • E (interpretation of data): The presence of cells stained with MHC dextramer, anti-CD3, anti-CD8 and not stained with anti-CD4 are T cells specific for Borrelia antigens and their frequency can optionally be determined as follows. The number of anti-CD8 and anti-CD3 stained cells and that of anti-CD8, MHC dextramer and anti-CD3 stained cells are determined. The frequency of specific MHC dextramer binding cells, or antigen specific T-cells towards Borrelia specific peptide is calculated as “# of MHC dextramer positive, CD3+, CD8+cells/total # of CD3+, CD8 positive cells”.
  • Process 10: Detection of Borrelia Specific T Cells in Sample of Purified Lymphocytes Derived from Human Blood
  • A (sampling): Use of vacutainer or other container containing anti-coagulant and cell separation medium to collect 4-10 ml human blood.
  • B (sample preparation): Vacutainer is centrifuged thereby separating cells according to size and lymphocytes are transferred to a new container. A fraction or all purified lymphocytes are added one or more MHC dextramers labeled with fluorochrome and carrying antigenic peptides derived from proteins from Borrelia bacteria. The MHC dextramers may all carry the same MHC I allele or carry different MHC I alleles.
  • Borrelia bacteria here include any strain of the species Borrelia Burgdorferi, Borrelia Garinii or Borrelia Afzelii. Example Borrelia proteins include but is not limited to OspA, OspB, OspC, OspE, OspG, VIsE, FlaB, FIaA, BmpA, BmpB, BmpC, BmpD, HSP90, GRASP-1, CRASP-2, DbpA, DbpB, MaIQ, BapA, other outer surface Borrelia proteins, other intracellular proteins of Borrelia and plasmid encoded proteins from Borrelia bacteria.
  • The antigenic peptides are preferably 9′mers but can also be 8′mers, 10′ers or 11′mers.
  • The sample and MHC dextramers are incubated for 1-30′ at room temperature in the dark, then gating reagents including labeled markers directed against CD3, CD8 and CD4 are added and incubation continued for 1-60′. The sample is centrifuged and supernatant removed. Cells are washed by resuspension in buffer followed by centrifugation. Supernatant is removed and cells resuspended in buffer.
  • Optionally fixative is added.
  • C (assaying): The prepared sample is applied to a flow cytometer and data acquired.
  • D (data analysis): The obtained data are analyzed for the presences of cells stained with MHC dextramer, anti-CD3, anti-CD8 and not stained with anti-CD4.
  • E (interpretation of data): The presence of cells stained with MHC dextramer, anti-CD3, anti-CD8 and not stained with anti-CD4 are T cells specific for Borrelia antigens and their frequency can optionally be determined as follows. The number of anti-CD8 and anti-CD3 stained cells and that of anti-CD8, MHC dextramer and anti-CD3 stained cells are determined. The frequency of specific MHC dextramer binding cells, or antigen specific T-cells towards Borrelia specific peptide is calculated as “# of MHC dextramer positive, CD3+, CD8+cells/total # of CD3+, CD8 positive cells”.
  • Process 11: Detection of Borrelia Specific T Cells in Sample of Human Blood Using a No-Lyse-No Wash Method.
  • A (sampling): Use of vacutainer or other container containing anti-coagulant to collect 4-10 ml human blood.
  • B (sample preparation): The blood is transferred to tube containing cell separation medium, e.g. ficoll. The tube is centrifuged thereby separating cells according to size and lymphocytes are transferred to a new container. A fraction or all purified lymphocytes are added one or more MHC dextramers labeled with fluorochrome and carrying antigenic peptides derived from proteins from Borrelia bacteria.
  • Borrelia bacteria here include any strain of the species Borrelia Burgdorferi, Borrelia Garinii or Borrelia Afzelii. Example Borrelia proteins include but is not limited to OspA, OspB, OspC, OspE, OspG, VIsE, FlaB, FIaA, BmpA, BmpB, BmpC, BmpD, HSP90, GRASP-1, CRASP-2, DbpA, DbpB, MaIQ, BapA, other outer surface Borrelia proteins, other intracellular proteins of Borrelia and plasmid encoded proteins from Borrelia bacteria.
  • The antigenic peptides are preferably 9′mers but can also be 8′mers, 10′ers or 11′mers.
  • The sample and MHC dextramers are incubated for 1-30′ at room temperature in the dark, then gating reagents including labeled markers directed against CD3, CD8 and CD4 are added and incubation continued for 1-60′.
  • Buffer is added to dilute sample 2-50 times.
  • Optionally the sample is added fixative.
  • C (assaying): The prepared sample is applied to a flow cytometer and data acquired.
  • D (data analysis): The obtained data are analyzed for the presences of cells stained with MHC dextramer, anti-CD3, anti-CD8 and not stained with anti-CD4.
  • E (interpretation of data): The presence of cells stained with MHC dextramer, anti-CD3, anti-CD8 and not stained with anti-CD4 are T cells specific for Borrelia antigens and their frequency can optionally be determined as follows. The number of anti-CD8 and anti-CD3 stained cells and that of anti-CD8, MHC dextramer and anti-CD3 stained cells are determined. The frequency of specific MHC dextramer binding cells, or antigen specific T-cells towards Borrelia specific peptide is calculated as “# of MHC dextramer positive, CD3+, CD8+cells/total # of CD3+, CD8 positive cells”.
  • Process 12: Detection of Borrelia Specific T Cells in Blood in a No-Lyse No-Wash Assay.
  • A (sampling): Use of vacutainer or other container containing anti-coagulant to collect 4-10 ml human blood.
  • B (sample preparation): 10-500 μl blood is transferred to a tube and added one or more MHC dextramers labeled with fluorochrome and carrying antigenic peptides derived from proteins from Borrelia bacteria.
  • Borrelia bacteria here include any strain of the species Borrelia Burgdorferi, Borrelia Garinii or Borrelia Afzelii. Example Borrelia proteins include but is not limited to OspA, OspB, OspC, OspE, OspG, VIsE, FlaB, FIaA, BmpA, BmpB, BmpC, BmpD, HSP90, GRASP-1, CRASP-2, DbpA, DbpB, MaIQ, BapA, other outer surface Borrelia proteins, other intracellular proteins of Borrelia and plasmid encoded proteins from Borrelia bacteria.
  • The antigenic peptides are preferably 9′mers but can also be 8′mers, 10′ers or 11′mers.
  • The sample and MHC dextramers are incubated for 1-30′ at room temperature in the dark, then gating reagents including labeled markers directed against CD3, CD8, CD4, CD14 and CD45 are added and incubation continued for 1-60′. Preferably markers are labeled with label different from MHC dextramer label and different from each other.
  • Anti-CD4 and anti-CD14 markers are “dump” gate markers and can be labeled with same fluorochrome. Other dump gate markers like anti-CD19, anti-CD15 may also be included in analysis and are then labeled with same label as anti-CD4/anti-CD14 and added to the sample together with these markers.
  • Buffer is added to dilute sample 2-50 times.
  • Optionally, an exact volume and known number of beads (counting beads), e.g. CytoCount beads can be added to the sample.
  • Optionally the sample is added fixative.
  • C (assaying): The prepared sample is applied to a flow cytometer and data acquired.
  • D (data analysis): The obtained data are analyzed for the presences of cells stained with MHC dextramer, anti-CD3, anti-CD8 as follows. Cells stained positive with anti-CD4 and anti-CD14 are deselected in a “dump” gate and remaining cells stained positive with anti-CD45 are selected for further analysis. In this population the presences of cells stained with MHC dextramer, anti-CD3 and anti-CD8 markers are determined.
  • The amount of acquired beads is also determined.
  • E (interpretation of data): The presence of cells stained with MHC dextramer, anti-CD3 and anti-CD8 are T cells specific for Borrelia antigens and their frequency can optionally be determined as follows. The number of anti-CD8 and anti-CD3 stained cells and that of anti-CD8, MHC dextramer and anti-CD3 stained cells are determined. The frequency of specific MHC dextramer binding cells, or antigen specific T-cells towards Borrelia specific peptide is calculated as “# of MHC dextramer positive, CD3+, CD8+cells/total # of CD3+, CD8 positive cells”.
  • If counting beads have been added the concentration of Borrelia specific T cells can be determined more accurately using the following calculations:

  • Concentration of Borrelia specific T cells=Dilution factor of blood×(# counted specific T cells×concentration of bead)/# counted beads.
  • Process 13: Detection of Borrelia Specific T Cells in Blood in a No-Lyse No-Wash Assay Using a Tube Containing Solidified Reagents.
  • A (sampling): Use of vacutainer or other tube containing anti-coagulant to collect 4-10 ml human blood.
  • B (sample preparation): 10-500 μl blood is transferred to a tube containing a solid or semisolid matrix featuring one or more MHC dextramers labeled with fluorochrome and carrying antigenic peptides derived from proteins from Borrelia bacteria.
  • Borrelia bacteria here include any strain of the species Borrelia Burgdorferi, Borrelia Garinii or Borrelia Afzelii. Example Borrelia proteins include but is not limited to OspA, OspB, OspC, OspE, OspG, VIsE, FlaB, FIaA, BmpA, BmpB, BmpC, BmpD, HSP90, GRASP-1, CRASP-2, DbpA, DbpB, MaIQ, BapA, other outer surface Borrelia proteins, other intracellular proteins of Borrelia and plasmid encoded proteins from Borrelia bacteria.
  • The antigenic peptides are preferably 9′mers but can also be 8′mers, 10′ers or 11′mers.
  • The matrix also features gating reagents including labeled markers directed against CD3, CD8, CD4, CD14 and CD45. Preferably markers are labeled with label different from MHC dextramer label and different from each other. Anti-CD4 and anti-CD14 markers are “dump” gate markers and can be labeled with same fluorochrome. Other dump-gate markers like anti-CD19, anti-CD15 or others may also be included in analysis and are then labeled with same label as anti-CD4/anti-CD14 markers and are also contained in matrix material.
  • The matrix material is such that MHC dextramers and gating reagents are released upon addition of blood. Preferably the matrix material is dissolved thereby releasing reagents into added sample.
  • The sample is incubated for 1-60′ at room temperature in the dark. Optionally additional gating reagents can be added. Buffer is added to dilute sample 2-50 times.
  • Optionally, an exact volume and known number of beads (counting beads), e.g. CytoCount beads can be added to the sample.
  • Optionally the sample is added fixative.
  • C (assaying): The prepared sample is applied to a flow cytometer and data acquired.
  • D (data analysis): The obtained data are analyzed for the presences of cells stained with MHC dextramer, anti-CD3, anti-CD8 as follows. Cells stained positive with anti-CD4 and anti-CD14 are deselected in a “dump” gate and remaining cells stained positive with anti-CD45 are selected for further analysis. In this population the presences of cells stained with MHC dextramer, anti-CD3 and anti-CD8 markers are determined.
  • The amount of acquired beads is also determined.
  • E (interpretation of data): The presence of cells stained with MHC dextramer, anti-CD3 and anti-CD8 are T cells specific for Borrelia antigens and their frequency can optionally be determined as follows. The number of anti-CD8 and anti-CD3 stained cells and that of anti-CD8, MHC dextramer and anti-CD3 stained cells are determined. The frequency of specific MHC dextramer binding cells, or antigen specific T-cells towards Borrelia specific peptide is calculated as “# of MHC dextramer positive, CD3+, CD8+cells/total # of CD3+, CD8 positive cells”.
  • If counting beads have been added the concentration of Borrelia specific T cells can be determined more accurately using the following calculations:

  • Concentration of Borrelia specific T cells=Dilution factor of blood×(# counted specific T cells×concentration of bead)/# counted beads.
  • Process 14: Determination of Absolute Count of CD3+, CD4+and CD3+, CD8+T Cell Subsets in Human Blood.
  • A (sampling): Use of vacutainer or other tube containing anti-coagulant to collect 4-10 ml human blood.
  • B (sample preparation): 10-500 μl blood is transferred to a tube together with anti-CD3, anti-CD4 and anti-CD8 markers labeled with different fluorochromes. The sample is incubated for 1-60′ at room temperature in the dark and lysis reagent is added, e.g. EasyLyse or iTAG MHC Tetramer Lyse reagent. Following incubation buffer is added to dilute sample 2-50 times.
  • Optionally the sample is added fixative.
  • An exact volume and known number of beads (counting beads), e.g. Flow-Count fluorospheres are added to the sample.
  • C (assaying): The prepared sample is applied to a flow cytometer and data acquired.
  • D (data analysis): The obtained data are analyzed for the presences of cells stained with anti-CD3 and anti-CD8 markers and cells stained with anti-CD3 and anti-CD4 markers.
  • The presence and amount of beads acquired is also analyzed.
  • E (interpretation of data): The number of anti-CD8+, anti-CD3 double positive cells and the number of anti-CD4+, anti-CD3+double positive cells acquired are determined.
  • The number of acquired beads is also determined. The absolute concentration of CD3+, CD8+positive cells can be calculated as follows:

  • Concentration of CD3+, CD8+positive cells=(# counted CD3+, CD8+positive cells×concentration of bead)/# counted beads.
  • Likewise the absolute concentration of CD3+, CD4+positive cells can be calculated as follows:

  • Concentration of CD3+, CD4+positive cells=(# counted CD3+, CD4+positive cells×concentration of bead)/# counted beads.
  • Process 15: Determination of Absolute Count of Borrelia Specific T Cells in Human Blood Using a Two-Panel Technique.
  • A (sampling): Use of vacutainer or other tube containing anti-coagulant to collect 4-10 ml human blood.
  • B (sample preparation): 10-500 μl blood is transferred to a two tubes. The first tube is added anti-CD3, anti-CD4 and anti-CD8 markers labeled with different fluorochromes either before or after addition of blood.
  • The second tube are added anti-CD3, anti-CD4 and anti-CD8 markers labeled with different fluorochromes and also contains one or more MHC dextramers labeled with fluorochrome and carrying antigenic peptides derived from proteins from Borrelia bacteria. The label of the individual MHC dextramers are either the same or different from each other. However all MHC dextramers are labeled with label(s) different for the labels used for the other marker molecules.
  • Borrelia bacteria here include any strain of the species Borrelia Burgdorferi, Borrelia Garinii or Borrelia Afzelii. Example Borrelia proteins include but is not limited to OspA, OspB, OspC, OspE, OspG, VIsE, FlaB, FIaA, BmpA, BmpB, BmpC, BmpD, HSP90, CRASP-1, CRASP-2, DbpA, DbpB, MaIQ, BapA, other outer surface Borrelia proteins, other intracellular proteins of Borrelia and plasmid encoded proteins from Borrelia bacteria.
  • The antigenic peptides are preferably 9′mers but can also be 8′mers, 10′ers or 11′mers.
  • Both tubes are incubated for 1-60′ at room temperature in the dark and lysis reagent is added, e.g. EasyLyse or iTAG MHC Tetramer Lyse reagent. The lysis reagent may optionally contain fixative.
  • Following incubation tube one is added buffer to dilute sample 0-50 times and an exact volume and known number of beads (counting beads), e.g. Flow-Count fluorospheres are added to tube one.
  • Tube two is centrifuged, supernatant removed and buffer added to wash cells.
  • Following centrifugation supernatant is removed and cells resuspended in buffer with or without fixative.
  • C (assaying): The prepared samples are applied to a flow cytometer and data acquired.
  • D (data analysis): The obtained data for tube 1 are analyzed for the presences of cells stained with anti-CD3 and anti-CD8 markers and cells stained with anti-CD3 and anti-CD4 markers. The presence and amount of beads acquired is also analyzed.
  • The obtained data for tube 2 are analyzed for the presence of cells stained with anti-CD3 and anti-CD8 markers, cells stained with anti-CD3 and anti-CD4 markers and also cells triple stained with MHC dextramers, anti-CD3 and anti-CD8 markers.
  • E (interpretation of data): The number of anti-CD8+, anti-CD3 double positive cells and the number of anti-CD4+, anti-CD3+double positive cells acquired in tube one and tube 2 are determined. The number of acquired beads in tube one is also determined.
  • The absolute concentration of CD3+, CD8+positive cells can be calculated as follows:

  • Concentration of CD3+, CD8+positive cells=(# counted CD3+, CD8+positive cells×concentration of bead)/# counted beads.
  • Likewise the absolute concentration of CD3+, CD4+positive cells can be calculated as follows: Concentration of CD3+, CD4+positive cells pr. volume blood=(# counted CD3+, CD4+positive cells×concentration of bead)/ # counted beads.
  • The frequency of specific MHC dextramer binding cells, or antigen specific T-cells towards Borrelia specific peptide is calculated as: (# of MHC dextramer positive, CD3+ and CD8+cells)/(total # of CD3+, CD8+positive cells).
  • Absolute concentration of Borrelia specific T cells can then be calculated as follows:

  • (Absolute concentration of CD3+, CD8+double positive cells)×(frequency of MHC dextramer+, CD3+, CD8+triple positive cells).
  • Process 16: Determination of Presences of Borrelia Specific T Cells in Human Blood Including a Negative Control Reagent.
  • A (sampling): Use of vacutainer or other container containing anti-coagulant to collect 4-10 ml human blood.
  • B (sample preparation): 10-500 μl of the full blood is transferred to one or more tubes containing one or more MHC dextramers labeled with fluorochrome and carrying antigenic peptides derived from proteins from Borrelia bacteria. MHC dextramers are MHC-peptide complexes e.g. generated by in vitro refolding, coupled to dextran through covalent or non-covalent interactions. The dextramer furthermore comprises fluorochrome coupled to the same dextran molecules through covalent or non-covalent interactions.
  • The MHC dextramers may all carry the same MHC I allele or carry different MHC I alleles.
  • Borrelia bacteria here include any strain of the species Borrelia Burgdorferi, Borrelia Garinii or Borrelia Afzelii. Example Borrelia proteins include but is not limited to OspA, OspB, OspC, OspE, OspG, VIsE, FlaB, FIaA, BmpA, BmpB, BmpC, BmpD, HSP90, GRASP-1, CRASP-2, DbpA, DbpB, MaIQ, BapA, other outer surface Borrelia proteins, other intracellular proteins of Borrelia and plasmid encoded proteins from Borrelia bacteria.
  • To another tube same volume of blood is added together with one or more MHC dextramer(s) labeled with fluorochrome and each carrying a negative control peptide. Negative control peptides are peptides binding the MHC allele of choice, but that does not support binding of the resultant MHC-peptide complex to the desired TCR.
  • Example negative control peptides include but are not limited to modified peptides, naturally occurring peptide different from the peptide used for analysis and nonsense peptides (peptides with an amino acid sequence different from the linear sequence of any peptide derived from any known protein).
  • The antigenic peptides and/or negative control peptides are preferably 9′mers but can also be 8′mers, 10′ers or 11′mers.
  • All samples are incubated for 1-60′ at room temperature in the dark, then gating reagents including labeled markers directed against CD3, CD8 and CD4 are added and incubation continued for 1-60′. RBC is lysed by addition of lysis reagents e.g. EasyLyse then the sample is centrifuged, supernatant removed.
  • Cells are washed by resuspension in buffer followed by centrifugation. Supernatant is removed and cells resuspended in buffer.
  • Optionally fixative is added.
  • C (assaying): The prepared samples are applied to a flow cytometer and data acquired.
  • D (data analysis): The obtained data are analyzed for the presences of cells stained with MHC dextramer, anti-CD3, anti-CD8 and not stained with anti-CD4.
  • E (interpretation of data): Data from samples stained with MHC dextramers carrying negative control peptides are used as negative controls to assess the level of background fluorescence and non-specific binding of all MHC dextramers.
  • The presence of cells triple stained with MHC dextramers containing borrelia peptides, anti-CD3, anti-CD8 and not stained with anti-CD4 are T cells specific for Borrelia antigens and their frequency can optionally be determined as follows. The number of anti-CD8 and anti-CD3 stained cells and that of anti-CD8, MHC dextramer and anti-CD3 stained cells are determined. The frequency of specific MHC dextramer binding cells, or antigen specific T-cells towards Borrelia specific peptide is calculated as “# of MHC dextramer positive, CD3+and CD8+triple positive cells/total # of CD3+,CD8+ double positive cells”.
  • Negative control reagents comprising MHC dextramers carrying negative control peptides can be included in all processes involving staining with MHC dextramers.
  • Process 17: Detection of Borrelia Specific T Cells in Human Blood Using MHC Dextramers Carrying MHC II Molecules
  • A (sampling): Use of vacutainer or other container containing anti-coagulant to collect 4-10 ml human blood.
  • B (sample preparation): 10-500 μl of the full blood is transferred to a tube containing one or more MHC dextramers labeled with fluorochrome and carrying antigenic peptides derived from proteins from Borrelia bacteria. MHC dextramers are MHC-peptide complexes e.g. generated by in vitro refolding, coupled to dextran through covalent or non-covalent interactions. The dextramer furthermore comprises fluorochrome coupled to the same dextran molecules through covalent or non-covalent interactions. The MHC dextramers may all carry the same MHC II allele or carry different MHC II alleles.
  • Borrelia bacteria here include any strain of the species Borrelia Burgdorferi, Borrelia Garinii or Borrelia Afzelii. Example Borrelia proteins include but is not limited to OspA, OspB, OspC, OspE, OspG, VIsE, FlaB, FIaA, BmpA, BmpB, BmpC, BmpD, HSP90, GRASP-1, CRASP-2, DbpA, DbpB, MaIQ, BapA, other outer surface Borrelia proteins, other intracellular proteins of Borrelia and plasmid encoded proteins from Borrelia bacteria.
  • The antigenic peptides are preferably 12-18′mers but can also be 9′mers, 10′ers, 11′mers, 19′mers, 20′mers or even longer peptide fragments.
  • The MHC dextramers are incubated for 1-60′ at room temperature in the dark, then gating reagents including labeled markers directed against CD3, CD8 and CD4 are added and incubation continued for 1-60′. RBC is lysed by addition of lysis reagents e.g. EasyLyse then the sample is centrifuged, supernatant removed.
  • Cells are washed by resuspension in buffer followed by centrifugation. Supernatant is removed and cells resuspended in buffer.
  • Optionally fixative is added.
  • C (assaying): The prepared sample is applied to a flow cytometer and data acquired.
  • D (data analysis): The obtained data are analyzed for the presences of cells stained with MHC dextramer, anti-CD3, anti-CD4 and not stained with anti-CD8.
  • E (interpretation of data): The presence of cells stained with MHC dextramer, anti-CD3, anti-CD4 and not stained with anti-CD8 are T cells specific for Borrelia antigens and their frequency can optionally be determined as follows. The number of anti-CD4 and anti-CD3 stained cells and that of anti-CD4, MHC dextramer and anti-CD3 stained cells are determined. The frequency of specific MHC dextramer binding cells, or antigen specific T-cells towards Borrelia specific peptide is calculated as “# of MHC dextramer positive, CD3+, CD4+cells/total # of CD3+, CD4 positive cells”.
  • Process 18: Detection of Borrelia Specific T Cells in Human Blood Using a Lyse No-Wash Method.
  • A (sampling): Use of vacutainer or other container containing anti-coagulant to collect 4-10 ml human blood.
  • B (sample preparation): 10-500 μl of the full blood is transferred to a tube containing one or more MHC dextramers labeled with fluorochrome and carrying antigenic peptides derived from proteins from Borrelia bacteria. MHC dextramers are MHC-peptide complexes e.g. generated by in vitro refolding, coupled to dextran through covalent or non-covalent interactions. The dextramer furthermore comprises fluorochrome coupled to the same dextran molecules through covalent or non-covalent interactions. The MHC dextramers may all carry the same MHC II allele or carry different MHC II alleles.
  • Borrelia bacteria here include any strain of the species Borrelia Burgdorferi, Borrelia Garinii or Borrelia Afzelii. Example Borrelia proteins include but is not limited to OspA, OspB, OspC, OspE, OspG, VIsE, FlaB, FIaA, BmpA, BmpB, BmpC, BmpD, HSP90, GRASP-1, CRASP-2, DbpA, DbpB, MaIQ, BapA, other outer surface Borrelia proteins, other intracellular proteins of Borrelia and plasmid encoded proteins from Borrelia bacteria.
  • The antigenic peptides are preferably 12-18′mers but can also be 9′mers, 10′ers, 11′mers, 19′mers, 20′mers or even longer peptide fragments.
  • The MHC dextramers are incubated for 1-30′ at room temperature in the dark, then gating reagents including labeled markers directed against CD3, CD8 and CD4 are added and incubation continued for 1-60′. RBC is lysed by addition of lysis reagents e.g. EasyLyse and buffer is added to dilute sample 2-50 times.
  • Optionally fixative is added.
  • C (assaying): The prepared sample is applied to a flow cytometer and data acquired.
  • D (data analysis): The obtained data are analyzed for the presences of cells stained with MHC dextramer, anti-CD3, anti-CD4 and not stained with anti-CD8.
  • E (interpretation of data): The presence of cells stained with MHC dextramer, anti-CD3, anti-CD4 and not stained with anti-CD8 are T cells specific for Borrelia antigens and their frequency can optionally be determined as follows. The number of anti-CD4 and anti-CD3 stained cells and that of anti-CD4, MHC dextramer and anti-CD3 stained cells are determined. The frequency of specific MHC dextramer binding cells, or antigen specific T-cells towards Borrelia specific peptide is calculated as “# of MHC dextramer positive, CD3+, CD4+cells/total # of CD3+, CD4 positive cells”.
  • Process 19: Detection of Borrelia Specific T Cells in Sample of Purified Lymphocytes Derived from Human Blood
  • A (sampling): Use of vacutainer or other container containing anti-coagulant and cell separation medium to collect 4-10 ml human blood.
  • B (sample preparation): Vacutainer is centrifuged thereby separating cells according to size and lymphocytes are transferred to a new container. A fraction or all purified lymphocytes are added one or more MHC dextramers labeled with fluorochrome and carrying antigenic peptides derived from proteins from Borrelia bacteria. The MHC dextramers may all carry the same MHC II allele or carry different MHC II alleles.
  • Borrelia bacteria here include any strain of the species Borrelia Burgdorferi, Borrelia Garinii or Borrelia Afzelii. Example Borrelia proteins include but is not limited to OspA, OspB, OspC, OspE, OspG, VIsE, FlaB, FIaA, BmpA, BmpB, BmpC, BmpD, HSP90, GRASP-1, CRASP-2, DbpA, DbpB, MaIQ, BapA, other outer surface Borrelia proteins, other intracellular proteins of Borrelia and plasmid encoded proteins from Borrelia bacteria.
  • The antigenic peptides are preferably 12-18′mers but can also be 9′mers, 10′ers, 11′mers, 19′mers, 20′mers or even longer peptide fragments.
  • The sample and MHC dextramers are incubated for 1-30′ at room temperature in the dark, then gating reagents including labeled markers directed against CD3, CD8 and CD4 are added and incubation continued for 1-60′. The sample is centrifuged and supernatant removed. Cells are washed by resuspension in buffer followed by centrifugation. Supernatant is removed and cells resuspended in buffer.
  • Optionally fixative is added.
  • C (assaying): The prepared sample is applied to a flow cytometer and data acquired.
  • D (data analysis): The obtained data are analyzed for the presences of cells stained with MHC dextramer, anti-CD3, anti-CD4 and not stained with anti-CD8.
  • E (interpretation of data): The presence of cells stained with MHC dextramer, anti-CD3, anti-CD4 and not stained with anti-CD8 are T cells specific for Borrelia antigens and their frequency can optionally be determined as follows. The number of anti-CD4 and anti-CD3 stained cells and that of anti-CD4, MHC dextramer and anti-CD3 stained cells are determined. The frequency of specific MHC dextramer binding cells, or antigen specific T-cells towards Borrelia specific peptide is calculated as “# of MHC dextramer positive, CD3+, CD4+cells/total # of CD3+, CD4 positive cells”.
  • Process 20: Detection of Borrelia Specific T Cells in Sample of Purified Lymphocytes Derived from Human Blood
  • A (sampling): Use of vacutainer or other container containing anti-coagulant to collect 4-10 ml human blood.
  • B (sample preparation): The blood is transferred to tube containing cell separation medium, e.g. ficoll. The tube is centrifuged thereby separating cells according to size and lymphocytes are transferred to a new container. A fraction or all purified lymphocytes are added one or more MHC dextramers labeled with fluorochrome and carrying antigenic peptides derived from proteins from Borrelia bacteria.
  • Borrelia bacteria here include any strain of the species Borrelia Burgdorferi, Borrelia Garinii or Borrelia Afzelii. Example Borrelia proteins include but is not limited to OspA, OspB, OspC, OspE, OspG, VIsE, FlaB, FIaA, BmpA, BmpB, BmpC, BmpD, HSP90, GRASP-1, CRASP-2, DbpA, DbpB, MaIQ, BapA, other outer surface Borrelia proteins, other intracellular proteins of Borrelia and plasmid encoded proteins from Borrelia bacteria.
  • The antigenic peptides are preferably 12-18′mers but can also be 9′mers, 10′ers, 11′mers, 19′mers, 20′mers or even longer peptide fragments.
  • The sample and MHC dextramers are incubated for 1-30′ at room temperature in the dark, then gating reagents including labeled markers directed against CD3, CD8 and
  • CD4 are added and incubation continued for 1-60′.
  • Buffer is added to dilute sample 2-50 times.
  • Optionally the sample is added fixative.
  • C (assaying): The prepared sample is applied to a flow cytometer and data acquired.
  • D (data analysis): The obtained data are analyzed for the presences of cells stained with MHC dextramer, anti-CD3, anti-CD4 and not stained with anti-CD8.
  • E (interpretation of data): The presence of cells stained with MHC dextramer, anti-CD3, anti-CD4 and not stained with anti-CD8 are T cells specific for Borrelia antigens and their frequency can optionally be determined as follows. The number of anti-CD4 and anti-CD3 stained cells and that of anti-CD4, MHC dextramer and anti-CD3 stained cells are determined. The frequency of specific MHC dextramer binding cells, or antigen specific T-cells towards Borrelia specific peptide is calculated as “# of MHC dextramer positive, CD3+, CD4+cells/total # of CD3+, CD4 positive cells”.
  • Process 21: Detection of Borrelia Specific T Cells in Blood in a No-Lyse No-Wash Assay.
  • A (sampling): Use of vacutainer or other container containing anti-coagulant to collect 4-10 ml human blood.
  • B (sample preparation): 10-500 μl blood is transferred to a tube and added one or more MHC dextramers labeled with fluorochrome and carrying antigenic peptides derived from proteins from Borrelia bacteria.
  • Borrelia bacteria here include any strain of the species Borrelia Burgdorferi, Borrelia Garinii or Borrelia Afzelii. Example Borrelia proteins include but is not limited to OspA, OspB, OspC, OspE, OspG, VIsE, FlaB, FIaA, BmpA, BmpB, BmpC, BmpD, HSP90, GRASP-1, CRASP-2, DbpA, DbpB, MaIQ, BapA, other outer surface Borrelia proteins, other intracellular proteins of Borrelia and plasmid encoded proteins from Borrelia bacteria.
  • The antigenic peptides are preferably 12-18′mers but can also be 9′mers, 10′ers, 11′mers, 19′mers, 20′mers or even longer peptide fragments.
  • The sample and MHC dextramers are incubated for 1-30′ at room temperature in the dark, then gating reagents including labeled markers directed against CD3, CD8, CD4, CD14 and CD45 are added and incubation continued for 1-60′. Preferably markers are labeled with label different from MHC dextramer label and different from each other.
  • Anti-CD8 and anti-CD14 markers are “dump” gate markers and can be labeled with same fluorochrome. Other dump gate markers like anti-CD19, anti-CD15 may also be included in analysis and are then labeled with same label as anti-CD8/anti-CD14 and added to the sample together with these markers.
  • Buffer is added to dilute sample 2-50 times.
  • Optionally, an exact volume and known number of beads (counting beads), e.g. CytoCount beads can be added to the sample.
  • Optionally the sample is added fixative.
  • C (assaying): The prepared sample is applied to a flow cytometer and data acquired.
  • D (data analysis): The obtained data are analyzed for the presences of cells stained with MHC dextramer, anti-CD3, anti-CD4 as follows. Cells stained positive with anti-CD8 and anti-CD14 are deselected in a “dump” gate and remaining cells stained positive with anti-CD45 are selected for further analysis. In this population the presences of cells stained with MHC dextramer, anti-CD3 and anti-CD4 markers are determined.
  • The amount of acquired beads is also determined.
  • E (interpretation of data): The presence of cells stained with MHC dextramer, anti-CD3 and anti-CD4 are T cells specific for Borrelia antigens and their frequency can optionally be determined as follows. The number of anti-CD4 and anti-CD3 stained cells and that of anti-CD4, MHC dextramer and anti-CD3 stained cells are determined.
  • The frequency of specific MHC dextramer binding cells, or antigen specific T-cells towards Borrelia specific peptide is calculated as “# of MHC dextramer positive, CD3+, CD4+cells/total # of CD3+, CD4 positive cells”.
  • If counting beads have been added the concentration of Borrelia specific T cells can be determined more accurately using the following calculations:

  • Concentration of Borrelia specific T cells=Dilution factor of blood×(# counted specific T cells×concentration of bead)/# counted beads.
  • Process 22: Determination of Absolute Count of Borrelia Specific T Cells in Human Blood Using a Two-Panel Technique.
  • A (sampling): Use of vacutainer or other tube containing anti-coagulant to collect 4-10 ml human blood.
  • B (sample preparation): 10-500 μl blood is transferred to a two tubes. The first tube is added anti-CD3, anti-CD4 and anti-CD8 markers labeled with different fluorochromes either before or after addition of blood.
  • The second tube are added anti-CD3, anti-CD4 and anti-CD8 markers labeled with different fluorochromes and also contains one or more MHC dextramers labeled with fluorochrome and carrying antigenic peptides derived from proteins from Borrelia bacteria. The label of the individual MHC dextramers are either the same or different from each other. However all MHC dextramers are labeled with label(s) different for the labels used for the other marker molecules.
  • Borrelia bacteria here include any strain of the species Borrelia Burgdorferi, Borrelia Garinii or Borrelia Afzelii. Example Borrelia proteins include but is not limited to OspA, OspB, OspC, OspE, OspG, VIsE, FlaB, FIaA, BmpA, BmpB, BmpC, BmpD, HSP90, CRASP-1, CRASP-2, DbpA, DbpB, MaIQ, BapA, other outer surface Borrelia proteins, other intracellular proteins of Borrelia and plasmid encoded proteins from Borrelia bacteria.
  • The antigenic peptides are preferably 12-18′mers but can also be 9′mers, 10′ers, 11′mers, 19′mers, 20′mers or even longer peptide fragments.
  • Both tubes are incubated for 1-60′ at room temperature in the dark and lysis reagent is added, e.g. EasyLyse or iTAG MHC Tetramer Lyse reagent. The lysis reagent may optionally contain fixative.
  • Following incubation tube one is added buffer to dilute sample 0-50 times and an exact volume and known number of beads (counting beads), e.g. Flow-Count fluorospheres are added to tube one.
  • Tube two is centrifuged, supernatant removed and buffer added to wash cells.
  • Following centrifugation supernatant is removed and cells resuspended in buffer with or without fixative.
  • C (assaying): The prepared samples are applied to a flow cytometer and data acquired.
  • D (data analysis): The obtained data for tube 1 are analyzed for the presences of cells stained with anti-CD3 and anti-CD8 markers and cells stained with anti-CD3 and anti-CD4 markers. The presence and amount of beads acquired is also analyzed.
  • The obtained data for tube 2 are analyzed for the presence of cells stained with anti-CD3 and anti-CD8 markers, cells stained with anti-CD3 and anti-CD4 markers and also cells triple stained with MHC dextramers, anti-CD3 and anti-CD4 markers.
  • E (interpretation of data): The number of anti-CD8+, anti-CD3 double positive cells and the number of anti-CD4+, anti-CD3+ double positive cells acquired in tube one and tube 2 are determined. The number of acquired beads in tube one is also determined. The absolute concentration of CD3+, CD8+ positive cells can be calculated as follows:

  • Concentration of CD3+, CD8+positive cells=(# counted CD3+, CD8+positive cells×concentration of bead)/# counted beads.
  • Likewise the absolute concentration of CD3+, CD4+positive cells can be calculated as follows: Concentration of CD3+, CD4+positive cells pr. volume blood=(# counted CD3+, CD4+positive cells×concentration of bead)/# counted beads.
  • The frequency of specific MHC dextramer binding cells, or antigen specific T-cells towards Borrelia specific peptide is calculated as: (# of MHC dextramer positive, CD3+ and CD4+ cells)/(total # of CD3+, CD4+ positive cells).
  • Absolute concentration of Borrelia specific T cells can then be calculated as follows:

  • (Absolute concentration of CD3+, CD4+double positive cells)×(frequency of MHC dextramer+, CD3+, CD4+triple positive cells).
  • Process 23: Detection of Borrelia Specific T Cells in Blood Using MHC Tetramers.
  • Borrelia specific T cells may be detected and enumerated using MHC tetramers carrying Borrelia specific peptides. MHC tetramers are Streptavidin or Avidin associated with 4 biotinylated MHC-peptide molecules and labeled with flourochrome e.g. APC or PE.
  • Preferred processes involving use of MHC tetramers carrying Borrelia specific peptides for detection of Borrelia specific T cells include processes similar to process 7-13, 15-22 but where MHC tetramers are used in stead of MHC dextramers.
  • Process 24: Detection of Borrelia Specific T Cells in Blood Using MHC Multimers.
  • Borrelia specific T cells may be detected and enumerated using MHC multimers carrying Borrelia specific peptides. MHC multimers are complexes comprising of more than one MHC-peptide complexes, held together by covalent or non-covalent interactions between a multimerization domain and one or more MHC-peptide complexes and where the multimerizations domain is a molecule, a complex of molecules, a cell or a solid support.
  • Preferred processes involving use of MHC multimers carrying Borrelia specific peptides for detection of Borrelia specific T cells include processes similar to process 7-13, 15-22 but where MHC multimers are used instead of MHC dextramers.
  • Process 25: Detection of CMV Specific T Cells in Human Blood.
  • A (sampling): Use of vacutainer or other container containing anti-coagulant to collect 4-10 ml human blood.
  • B (sample preparation): 100 μl of the full blood is transferred to a tube containing one or more MHC dextramers labeled with APC and carrying antigenic peptides derived from proteins from human Cytomegalovirus (CMV).
  • The MHC dextramers may all carry the same MHC I allele or carry different MHC I alleles.
  • Example CMV proteins include but is not limited to Matrix phosphoprotein 65 (pp65), DNA polymerase, immediate early protein 1 (IE1), immediate early protein 3 (IE3), any intracellular protein, any surface protein or any other protein from which peptide epitopes are presented by MHC I molecules.
  • The antigenic peptides are preferably 9′mers but can also be 8′mers, 10′ers or 11′mers. Example of antigenic peptides include but is not limited to NLVPMVATV binding the MHC I allele HLA-A*0201, VTEHDTLLY binding HLA-A*0101, QYDPVAALF binding HLA-A*2402, TPRVTGGGAM binding B*0702, ELRRKMMYM binding B*0801, IPSINVHHY binding
  • B*3501, AYAQKIFKI binding HLA-A*2402, KLGGALQAK binding HLA-A*0301, VYALPLKML binding HLA-A*2402 and RPHERNGFTVL binding HLA-B*0702.
  • The MHC dextramers are incubated for 10′ at room temperature in the dark, then gating reagents including anti-CD3/PE, anti-CD8/PB and anti-CD4/FITC are added and incubation continued for 20′. RBC is lysed by addition of lysis reagents e.g. EasyLyse then the sample is centrifuged, supernatant removed. Cells are washed by resuspension in buffer followed by centrifugation. Supernatant is removed and cells resuspended in buffer.
  • Optionally fixative is added.
  • C (assaying): The prepared sample is applied to a flow cytometer and data acquired.
  • D (data analysis): The obtained data are analyzed for the presences of cells stained with MHC dextramer, anti-CD3, anti-CD8 and not stained with anti-CD4.
  • E (interpretation of data): The presence of cells stained with MHC dextramer, anti-CD3, anti-CD8 and not stained with anti-CD4 are T cells specific for CMV antigens and their frequency can optionally be determined as follows. The number of anti-CD8 and anti-CD3 stained cells and that of anti-CD8, MHC dextramer and anti-CD3 stained cells are determined. The frequency of specific MHC dextramer binding cells, or antigen specific T-cells towards CMV specific peptide is calculated as “# of MHC dextramer positive, CD3+, CD8+ cells/total # of CD3+,CD8 positive cells”.
  • Process 26: Detection of CMV Specific T Cells in Human Blood Using MHC Dextramers
  • A (sampling): Use of vacutainer or other container containing anti-coagulant to collect 4-10 ml human blood.
  • B (sample preparation): 10-500 μl of the full blood is transferred to a tube containing one or more MHC dextramers labeled with fluorochrome and carrying antigenic peptides derived from proteins from CMV.
  • The MHC dextramers may all carry the same MHC I allele or carry different MHC I alleles.
  • Example CMV proteins include but is not limited to Matrix phosphoprotein 65 (pp65), DNA polymerase, immediate early protein 1 (IE1), immediate early protein 3 (IE3), any intracellular protein, any surface protein or any other protein from which peptide epitopes are presented by MHC I molecules.
  • The antigenic peptides are preferably 9′mers but can also be 8′mers, 10′ers or 11′mers. Example of antigenic peptides include but is not limited to NLVPMVATV binding the MHC I allele HLA-A*0201, VTEHDTLLY binding HLA-A*0101, QYDPVAALF binding HLA-A*2402, TPRVTGGGAM binding B*0702, ELRRKMMYM binding B*0801, IPSINVHHY binding B*3501, AYAQKIFKI binding HLA-A*2402, KLGGALQAK binding HLA-A*0301, VYALPLKML binding HLA-A*2402 and RPHERNGFTVL binding HLA-B*0702.
  • The MHC dextramers are incubated for 1-60′ at room temperature in the dark, then gating reagents including labeled markers directed against CD3, CD8 and CD4 are added and incubation continued for 1-60′. RBC is lysed by addition of lysis reagents e.g. EasyLyse then the sample is centrifuged, supernatant removed.
  • Cells are washed by resuspension in buffer followed by centrifugation. Supernatant is removed and cells resuspended in buffer.
  • Optionally fixative is added.
  • C (assaying): The prepared sample is applied to a flow cytometer and data acquired.
  • D (data analysis): The obtained data are analyzed for the presences of cells stained with MHC dextramer, anti-CD3, anti-CD8 and not stained with anti-CD4.
  • E (interpretation of data): The presence of cells stained with MHC dextramer, anti-CD3, anti-CD8 and not stained with anti-CD4 are T cells specific for CMV antigens and their frequency can optionally be determined as follows. The number of anti-CD8 and anti-CD3 stained cells and that of anti-CD8, MHC dextramer and anti-CD3 stained cells are determined. The frequency of specific MHC dextramer binding cells, or antigen specific T-cells towards CMV specific peptide is calculated as “# of MHC dextramer positive, CD3+, CD8+ cells/total # of CD3+, CD8 positive cells”.
  • Process 27: Detection of CMV Specific T Cells in Human Blood Using a Lyse No-Wash Method.
  • A (sampling): Use of vacutainer or other container containing anti-coagulant to collect 4-10 ml human blood.
  • B (sample preparation): 10-500 μl of the full blood is transferred to a tube containing one or more MHC dextramers labeled with fluorochrome and carrying antigenic peptides derived from proteins from CMV.
  • The MHC dextramers may all carry the same MHC I allele or carry different MHC I alleles.
  • Example CMV proteins include but is not limited to Matrix phosphoprotein 65 (pp65), DNA polymerase, immediate early protein 1 (IE1), immediate early protein 3 (IE3), any intracellular protein, any surface protein or any other protein from which peptide epitopes are presented by MHC I molecules.
  • The antigenic peptides are preferably 9′mers but can also be 8′mers, 10′ers or 11′mers. Example of antigenic peptides include but is not limited to NLVPMVATV binding the MHC I allele HLA-A*0201, VTEHDTLLY binding HLA-A*0101, QYDPVAALF binding HLA-A*2402, TPRVTGGGAM binding B*0702, ELRRKMMYM binding B*0801, IPSINVHHY binding B*3501, AYAQKIFKI binding HLA-A*2402, KLGGALQAK binding HLA-A*0301, VYALPLKML binding HLA-A*2402 and RPHERNGFTVL binding HLA-B*0702.
  • The MHC dextramers are incubated for 1-30′ at room temperature in the dark, then gating reagents including labeled markers directed against CD3, CD8 and CD4 are added and incubation continued for 1-60′. RBC is lysed by addition of lysis reagents e.g. EasyLyse and buffer is added to dilute sample 2-50 times.
  • Optionally fixative is added.
  • C (assaying): The prepared sample is applied to a flow cytometer and data acquired.
  • D (data analysis): The obtained data are analyzed for the presences of cells stained with MHC dextramer, anti-CD3, anti-CD8 and not stained with anti-CD4.
  • E (interpretation of data): The presence of cells stained with MHC dextramer, anti-CD3, anti-CD8 and not stained with anti-CD4 are T cells specific for CMV antigens and their frequency can optionally be determined as follows. The number of anti-CD8 and anti-CD3 stained cells and that of anti-CD8, MHC dextramer and anti-CD3 stained cells are determined. The frequency of specific MHC dextramer binding cells, or antigen specific T-cells towards CMV specific peptide is calculated as “# of MHC dextramer positive, CD3+, CD8+ cells/total # of CD3+, CD8 positive cells”.
  • Process 28: Detection of CMV Specific T Cells in Sample of Purified Lymphocytes Derived from Human Blood
  • A (sampling): Use of vacutainer or other container containing anti-coagulant and cell separation medium to collect 4-10 ml human blood.
  • B (sample preparation): Vacutainer is centrifuged thereby separating cells according to size and lymphocytes are transferred to a new container. A fraction or all purified lymphocytes are added one or more MHC dextramers labeled with fluorochrome and carrying antigenic peptides derived from proteins from CMV. The MHC dextramers may all carry the same MHC I allele or carry different MHC I alleles.
  • Example CMV proteins include but is not limited to Matrix phosphoprotein 65 (pp65), DNA polymerase, immediate early protein 1 (IE1), immediate early protein 3 (IE3), any intracellular protein, any surface protein or any other protein from which peptide epitopes are presented by MHC I molecules.
  • The antigenic peptides are preferably 9′mers but can also be 8′mers, 10′ers or 11′mers. Example of antigenic peptides include but is not limited to NLVPMVATV binding the MHC I allele HLA-A*0201, VTEHDTLLY binding HLA-A*0101, QYDPVAALF binding HLA-A*2402, TPRVTGGGAM binding B*0702, ELRRKMMYM binding B*0801, IPSINVHHY binding
  • B*3501, AYAQKIFKI binding HLA-A*2402, KLGGALQAK binding HLA-A*0301, VYALPLKML binding HLA-A*2402 and RPHERNGFTVL binding HLA-B*0702.
  • The sample and MHC dextramers are incubated for 1-30′ at room temperature in the dark, then gating reagents including labeled markers directed against CD3, CD8 and CD4 are added and incubation continued for 1-60′. The sample is centrifuged and supernatant removed. Cells are washed by resuspension in buffer followed by centrifugation. Supernatant is removed and cells resuspended in buffer.
  • Optionally fixative is added.
  • C (assaying): The prepared sample is applied to a flow cytometer and data acquired.
  • D (data analysis): The obtained data are analyzed for the presences of cells stained with MHC dextramer, anti-CD3, anti-CD8 and not stained with anti-CD4.
  • E (interpretation of data): The presence of cells stained with MHC dextramer, anti-CD3, anti-CD8 and not stained with anti-CD4 are T cells specific for CMV antigens and their frequency can optionally be determined as follows. The number of anti-CD8 and anti-CD3 stained cells and that of anti-CD8, MHC dextramer and anti-CD3 stained cells are determined. The frequency of specific MHC dextramer binding cells, or antigen specific T-cells towards CMV specific peptide is calculated as “# of MHC dextramer positive, CD3+, CD8+ cells/total # of CD3+, CD8 positive cells”.
  • Process 29: Detection of CMV Specific T Cells in Sample of Human Blood Using a No-Lyse-No Wash Method.
  • A (sampling): Use of vacutainer or other container containing anti-coagulant to collect 4-10 ml human blood.
  • B (sample preparation): The blood is transferred to tube containing cell separation medium, e.g. ficoll. The tube is centrifuged thereby separating cells according to size and lymphocytes are transferred to a new container. A fraction or all purified lymphocytes are added one or more MHC dextramers labeled with fluorochrome and carrying antigenic peptides derived from proteins from CMV.
  • Example CMV proteins include but is not limited to Matrix phosphoprotein 65 (pp65), DNA polymerase, immediate early protein 1 (IE1), immediate early protein 3 (IE3), any intracellular protein, any surface protein or any other protein from which peptide epitopes are presented by MHC I molecules.
  • The antigenic peptides are preferably 9′mers but can also be 8′mers, 10′ers or 11′mers. Example of antigenic peptides include but is not limited to NLVPMVATV binding the MHC I allele HLA-A*0201, VTEHDTLLY binding HLA-A*0101, QYDPVAALF binding HLA-A*2402, TPRVTGGGAM binding B*0702, ELRRKMMYM binding B*0801, IPSINVHHY binding B*3501, AYAQKIFKI binding HLA-A*2402, KLGGALQAK binding HLA-A*0301, VYALPLKML binding HLA-A*2402 and RPHERNGFTVL binding HLA-B*0702.
  • The sample and MHC dextramers are incubated for 1-30′ at room temperature in the dark, then gating reagents including labeled markers directed against CD3, CD8 and CD4 are added and incubation continued for 1-60′.
  • Buffer is added to dilute sample 2-50 times.
  • Optionally the sample is added fixative.
  • C (assaying): The prepared sample is applied to a flow cytometer and data acquired.
  • D (data analysis): The obtained data are analyzed for the presences of cells stained with MHC dextramer, anti-CD3, anti-CD8 and not stained with anti-CD4.
  • E (interpretation of data): The presence of cells stained with MHC dextramer, anti-CD3, anti-CD8 and not stained with anti-CD4 are T cells specific for CMV antigens and their frequency can optionally be determined as follows. The number of anti-CD8 and anti-CD3 stained cells and that of anti-CD8, MHC dextramer and anti-CD3 stained cells are determined. The frequency of specific MHC dextramer binding cells, or antigen specific T-cells towards CMV specific peptide is calculated as “# of MHC dextramer positive, CD3+, CD8+ cells/total # of CD3+, CD8 positive cells”.
  • Process 30: Detection of CMV Specific T Cells in Blood in a No-Lyse No-Wash Assay.
  • A (sampling): Use of vacutainer or other container containing anti-coagulant to collect 4-10 ml human blood.
  • B (sample preparation): 10-500 μl blood is transferred to a tube and added one or more MHC dextramers labeled with fluorochrome and carrying antigenic peptides derived from proteins from CMV.
  • Example CMV proteins include but is not limited to Matrix phosphoprotein 65 (pp65), DNA polymerase, immediate early protein 1 (IE1), immediate early protein 3 (IE3), any intracellular protein, any surface protein or any other protein from which peptide epitopes are presented by MHC I molecules.
  • The antigenic peptides are preferably 9′mers but can also be 8′mers, 10′ers or 11′mers. Example of antigenic peptides include but is not limited to NLVPMVATV binding the MHC I allele HLA-A*0201, VTEHDTLLY binding HLA-A*0101, QYDPVAALF binding HLA-A*2402, TPRVTGGGAM binding B*0702, ELRRKMMYM binding B*0801, IPSINVHHY binding B*3501, AYAQKIFKI binding HLA-A*2402, KLGGALQAK binding HLA-A*0301, VYALPLKML binding HLA-A*2402 and RPHERNGFTVL binding HLA-B*0702.
  • The sample and MHC dextramers are incubated for 1-30′ at room temperature in the dark, then gating reagents including labeled markers directed against CD3, CD8, CD4, CD14 and CD45 are added and incubation continued for 1-60′. Preferably markers are labeled with label different from MHC dextramer label and different from each other.
  • Anti-CD4 and anti-CD14 markers are “dump” gate markers and can be labeled with same fluorochrome. Other dump gate markers like anti-CD19, anti-CD15 may also be included in analysis and are then labeled with same label as anti-CD4/anti-CD14 and added to the sample together with these markers.
  • Buffer is added to dilute sample 2-50 times.
  • Optionally, an exact volume and known number of beads (counting beads), e.g. CytoCount beads can be added to the sample.
  • Optionally the sample is added fixative.
  • C (assaying): The prepared sample is applied to a flow cytometer and data acquired.
  • D (data analysis): The obtained data are analyzed for the presences of cells stained with MHC dextramer, anti-CD3, anti-CD8 as follows. Cells stained positive with anti-CD4 and anti-CD14 are deselected in a “dump” gate and remaining cells stained positive with anti-CD45 are selected for further analysis. In this population the presences of cells stained with MHC dextramer, anti-CD3 and anti-CD8 markers are determined.
  • The amount of acquired beads is also determined.
  • E (interpretation of data): The presence of cells stained with MHC dextramer, anti-CD3 and anti-CD8 are T cells specific for CMV antigens and their frequency can optionally be determined as follows. The number of anti-CD8 and anti-CD3 stained cells and that of anti-CD8, MHC dextramer and anti-CD3 stained cells are determined. The frequency of specific MHC dextramer binding cells, or antigen specific T-cells towards CMV specific peptide is calculated as “# of MHC dextramer positive, CD3+, CD8+ cells/total # of CD3+, CD8 positive cells”.
  • If counting beads have been added the concentration of CMV specific T cells can be determined more accurately using the following calculations:

  • Concentration of CMV specific T cells=Dilution factor of blood×(# counted specific T cells×concentration of bead)/# counted beads.
  • Process 31: Detection of CMV Specific T Cells in Blood in a No-Lyse No-Wash Assay Using a Tube Containing Solidified Reagents.
  • A (sampling): Use of vacutainer or other tube containing anti-coagulant to collect 4-10 ml human blood.
  • B (sample preparation): 10-500 μl blood is transferred to a tube containing a solid or semisolid matrix featuring one or more MHC dextramers labeled with fluorochrome and carrying antigenic peptides derived from proteins from CMV bacteria.
  • Example CMV proteins include but is not limited to Matrix phosphoprotein 65 (pp65), DNA polymerase, immediate early protein 1 (IE1), immediate early protein 3 (IE3), any intracellular protein, any surface protein or any other protein from which peptide epitopes are presented by MHC I molecules.
  • The antigenic peptides are preferably 9′mers but can also be 8′mers, 10′ers or 11′mers. Example of antigenic peptides include but is not limited to NLVPMVATV binding the MHC I allele HLA-A*0201, VTEHDTLLY binding HLA-A*0101, QYDPVAALF binding HLA-A*2402, TPRVTGGGAM binding B*0702, ELRRKMMYM binding B*0801, IPSINVHHY binding B*3501, AYAQKIFKI binding HLA-A*2402, KLGGALQAK binding HLA-A*0301, VYALPLKML binding HLA-A*2402 and RPHERNGFTVL binding HLA-B*0702.
  • The matrix also features gating reagents including labeled markers directed against CD3, CD8, CD4, CD14 and CD45. Preferably markers are labeled with label different from MHC dextramer label and different from each other. Anti-CD4 and anti-CD14 markers are “dump” gate markers and can be labeled with same fluorochrome. Other dump-gate markers like anti-CD19, anti-CD15 or others may also be included in analysis and are then labeled with same label as anti-CD4/anti-CD14 markers and are also contained in matrix material.
  • The matrix material is such that MHC dextramers and gating reagents are released upon addition of blood. Preferably the matrix material is dissolved thereby releasing reagents into added sample.
  • The sample is incubated for 1-60′ at room temperature in the dark. Optionally additional gating reagents can be added. Buffer is added to dilute sample 2-50 times.
  • Optionally, an exact volume and known number of beads (counting beads), e.g. CytoCount beads can be added to the sample.
  • Optionally the sample is added fixative.
  • C (assaying): The prepared sample is applied to a flow cytometer and data acquired.
  • D (data analysis): The obtained data are analyzed for the presences of cells stained with MHC dextramer, anti-CD3, anti-CD8 as follows. Cells stained positive with anti-CD4 and anti-CD14 are deselected in a “dump” gate and remaining cells stained positive with anti-CD45 are selected for further analysis. In this population the presences of cells stained with MHC dextramer, anti-CD3 and anti-CD8 markers are determined.
  • The amount of acquired beads is also determined.
  • E (interpretation of data): The presence of cells stained with MHC dextramer, anti-CD3 and anti-CD8 are T cells specific for CMV antigens and their frequency can optionally be determined as follows. The number of anti-CD8 and anti-CD3 stained cells and that of anti-CD8, MHC dextramer and anti-CD3 stained cells are determined. The frequency of specific MHC dextramer binding cells, or antigen specific T-cells towards CMV specific peptide is calculated as “# of MHC dextramer positive, CD3+, CD8+ cells/total # of CD3+, CD8 positive cells”.
  • If counting beads have been added the concentration of CMV specific T cells can be determined more accurately using the following calculations:

  • Concentration of CMV specific T cells=Dilution factor of blood×(# counted specific T cells×concentration of bead)/# counted beads.
  • Process 32: Determination of Absolute Count of CMV Specific T Cells in Human Blood Using a Two-Panel Technique.
  • A (sampling): Use of vacutainer or other tube containing anti-coagulant to collect 4-10 ml human blood.
  • B (sample preparation): 10-500 μl blood is transferred to a two tubes. The first tube is added anti-CD3, anti-CD4 and anti-CD8 markers labeled with different fluorochromes either before or after addition of blood.
  • The second tube are added anti-CD3, anti-CD4 and anti-CD8 markers labeled with different fluorochromes and also contains one or more MHC dextramers labeled with fluorochrome and carrying antigenic peptides derived from proteins from CMV bacteria.
  • The label of the individual MHC dextramers are either the same or different from each other. However all MHC dextramers are labeled with label(s) different for the labels used for the other marker molecules.
  • Example CMV proteins include but is not limited to Matrix phosphoprotein 65 (pp65), DNA polymerase, immediate early protein 1 (IE1), immediate early protein 3 (IE3), any intracellular protein, any surface protein or any other protein from which peptide epitopes are presented by MHC I molecules.
  • The antigenic peptides are preferably 9′mers but can also be 8′mers, 10′ers or 11′mers. Example of antigenic peptides include but is not limited to NLVPMVATV binding the MHC I allele HLA-A*0201, VTEHDTLLY binding HLA-A*0101, QYDPVAALF binding HLA-A*2402, TPRVTGGGAM binding B*0702, ELRRKMMYM binding B*0801, IPSINVHHY binding B*3501, AYAQKIFKI binding HLA-A*2402, KLGGALQAK binding HLA-A*0301, VYALPLKML binding HLA-A*2402 and RPHERNGFTVL binding HLA-B*0702.
  • Both tubes are incubated for 1-60′ at room temperature in the dark and lysis reagent is added, e.g. EasyLyse or iTAG MHC Tetramer Lyse reagent. The lysis reagent may optionally contain fixative.
  • Following incubation tube one is added buffer to dilute sample 0-50 times and an exact volume and known number of beads (counting beads), e.g. Flow-Count fluorospheres are added to tube one.
  • Tube two is centrifuged, supernatant removed and buffer added to wash cells. Following centrifugation supernatant is removed and cells resuspended in buffer with or without fixative.
  • C (assaying): The prepared samples are applied to a flow cytometer and data acquired.
  • D (data analysis): The obtained data for tube 1 are analyzed for the presences of cells stained with anti-CD3 and anti-CD8 markers and cells stained with anti-CD3 and anti-CD4 markers. The presence and amount of beads acquired is also analyzed.
  • The obtained data for tube 2 are analyzed for the presence of cells stained with anti-CD3 and anti-CD8 markers, cells stained with anti-CD3 and anti-CD4 markers and also cells triple stained with MHC dextramers, anti-CD3 and anti-CD8 markers.
  • E (interpretation of data): The number of anti-CD8+, anti-CD3 double positive cells and the number of anti-CD4+, anti-CD3+double positive cells acquired in tube one and tube 2 are determined. The number of acquired beads in tube one is also determined. The absolute concentration of CD3+, CD8+positive cells can be calculated as follows:

  • Concentration of CD3+, CD8+positive cells=(# counted CD3+, CD8+ positive cells×concentration of bead)/# counted beads.
  • Likewise the absolute concentration of CD3+, CD4+ positive cells can be calculated as follows: Concentration of CD3+, CD4+ positive cells pr. volume blood=(# counted CD3+, CD4+positive cells×concentration of bead)/# counted beads.
  • The frequency of specific MHC dextramer binding cells, or antigen specific T-cells towards CMV specific peptide is calculated as: (# of MHC dextramer positive, CD3+ and CD8+ cells)/(total # of CD3+, CD8+ positive cells).
  • Absolute concentration of CMV specific T cells can then be calculated as follows:

  • (Absolute concentration of CD3+, CD8+ double positive cells)×(frequency of MHC dextramer+, CD3+, CD8+triple positive cells).
  • Process 33: Determination of Presences of CMV Specific T Cells in Human Blood Including a Negative Control Reagent.
  • A (sampling): Use of vacutainer or other container containing anti-coagulant to collect 4-10 ml human blood.
  • B (sample preparation): 10-500 μl of the full blood is transferred to one or more tubes containing one or more MHC dextramers labeled with fluorochrome and carrying antigenic peptides derived from proteins from CMV. MHC dextramers are MHC-peptide complexes e.g. generated by in vitro refolding, coupled to dextran through covalent or non-covalent interactions. The dextramer furthermore comprises fluorochrome coupled to the same dextran molecules through covalent or non-covalent interactions.
  • The MHC dextramers may all carry the same MHC I allele or carry different MHC I alleles.
  • Example CMV proteins include but is not limited to Matrix phosphoprotein 65 (pp65),
  • DNA polymerase, immediate early protein 1 (IE1), immediate early protein 3 (IE3), any intracellular protein, any surface protein or any other protein from which peptide epitopes are presented by MHC I molecules.
  • The antigenic peptides are preferably 9′mers but can also be 8′mers, 10′ers or 11′mers. Example of antigenic peptides include but is not limited to NLVPMVATV binding the MHC I allele HLA-A*0201, VTEHDTLLY binding HLA-A*0101, QYDPVAALF binding HLA-A*2402, TPRVTGGGAM binding B*0702, ELRRKMMYM binding B*0801, IPSINVHHY binding B*3501, AYAQKIFKI binding HLA-A*2402, KLGGALQAK binding HLA-A*0301, VYALPLKML binding HLA-A*2402 and RPHERNGFTVL binding HLA-B*0702.
  • To another tube same volume of blood is added together with one or more MHC dextramer(s) labeled with fluorochrome and each carrying a negative control peptide. Negative control peptides are peptides binding the MHC allele of choice, but that does not support binding of the resultant MHC-peptide complex to the desired TCR. Example negative control peptides include but are not limited to modified peptides, naturally occurring peptide different from the peptide used for analysis and nonsense peptides (peptides with an amino acid sequence different from the linear sequence of any peptide derived from any known protein).
  • The antigenic peptides and/or negative control peptides are preferably 9′mers but can also be 8′mers, 10′ers or 11′mers.
  • All samples are incubated for 1-60′ at room temperature in the dark, then gating reagents including labeled markers directed against CD3, CD8 and CD4 are added and incubation continued for 1-60′. RBC is lysed by addition of lysis reagents e.g. EasyLyse then the sample is centrifuged, supernatant removed.
  • Cells are washed by resuspension in buffer followed by centrifugation. Supernatant is removed and cells resuspended in buffer.
  • Optionally fixative is added.
  • C (assaying): The prepared samples are applied to a flow cytometer and data acquired.
  • D (data analysis): The obtained data are analyzed for the presences of cells stained with MHC dextramer, anti-CD3, anti-CD8 and not stained with anti-CD4.
  • E (interpretation of data): Data from samples stained with MHC dextramers carrying negative control peptides are used as negative controls to assess the level of background fluorescence and non-specific binding of all MHC dextramers.
  • The presence of cells triple stained with MHC dextramers containing CMV peptides, anti-CD3, anti-CD8 and not stained with anti-CD4 are T cells specific for CMV antigens and their frequency can optionally be determined as follows. The number of anti-CD8 and anti-CD3 stained cells and that of anti-CD8, MHC dextramer and anti-CD3 stained cells are determined. The frequency of specific MHC dextramer binding cells, or antigen specific T-cells towards CMV specific peptide is calculated as “# of MHC dextramer positive, CD3+and CD8+ triple positive cells/total # of CD3+,CD8+ double positive cells”.
  • Negative control reagents comprising MHC dextramers carrying negative control peptides can be included in all processes involving staining with MHC dextramers.
  • Process 34: Detection of CMV Specific T Cells in Blood Using MHC Tetramers.
  • CMV specific T cells may be detected and enumerated using MHC tetramers carrying CMV specific peptides. MHC tetramers are Streptavidin or Avidin associated with 4 biotinylated MHC-peptide molecules and labeled with flourochrome e.g. APC or PE.
  • Preferred processes involving use of MHC tetramers carrying CMV specific peptides for detection of CMV specific T cells include processes similar to process 25-33 but where MHC tetramers are used in stead of MHC dextramers.
  • Process 35: Detection of CMV Specific T Cells in Blood Using MHC Multimers.
  • CMV specific T cells may be detected and enumerated using MHC multimers carrying CMV specific peptides. MHC multimers are complexes comprising of more than one MHC-peptide complexes, held together by covalent or non-covalent interactions between a multimerization domain and one or more MHC-peptide complexes and where the multimerizations domain is a molecule, a complex of molecules, a cell or a solid support.
  • Preferred processes involving use of MHC multimers carrying CMV specific peptides for detection of CMV specific T cells include processes similar to process 25-33 but where MHC multimers are used instead of MHC dextramers.
  • Process 36: Detection of TB Specific T Cells in Human Blood.
  • A (sampling): Use of vacutainer or other container containing anti-coagulant to collect 4-10 ml human blood.
  • B (sample preparation): 100 μl of the full blood is transferred to a tube containing one or more MHC dextramers labeled with APC and carrying antigenic peptides derived from proteins from Mycobacteria Tuberculosis (TB).
  • The MHC dextramers may all carry the same MHC I allele or carry different MHC I alleles.
  • During acute infection TB specific activated T cells will be present in increased amounts in an activated state compared to in healthy individuals. Also in individuals with latent TB specific T cells will be present. The presences of activated TB specific T cells may thereby act as a surrogate marker for active and/or latent infection with Mycobacterium tuberculosis. MHC multimers carrying TB specific peptides is in this example used to detect the presence of active and or latent TB specific T cells in the blood of patients infected with Mycobacterium tuberculosis.
  • Example TB proteins include but is not limited to Ag85b, Mtb39, CFP10, ESAT-6, Mtb8.4, Mtb9.9A, EsxG, any TB surface protein, any TB intracellular protein or any other TB protein from which peptide epitopes are presented by MHC I molecules.
  • The antigenic peptides are preferably 9′mers but can also be 8′mers, 10′ers or 11′mers.
  • The MHC dextramers are incubated for 10′ at room temperature in the dark, then gating reagents including anti-CD3/PE, anti-CD8/PB and anti-CD4/FITC are added and incubation continued for 20′. RBC is lysed by addition of lysis reagents e.g. EasyLyse then the sample is centrifuged, supernatant removed. Cells are washed by resuspension in buffer followed by centrifugation. Supernatant is removed and cells resuspended in buffer.
  • Optionally fixative is added.
  • C (assaying): The prepared sample is applied to a flow cytometer and data acquired.
  • D (data analysis): The obtained data are analyzed for the presences of cells stained with MHC dextramer, anti-CD3, anti-CD8 and not stained with anti-CD4.
  • E (interpretation of data): The presence of cells stained with MHC dextramer, anti-CD3, anti-CD8 and not stained with anti-CD4 are T cells specific for TB antigens and their frequency can optionally be determined as follows. The number of anti-CD8 and anti-CD3 stained cells and that of anti-CD8, MHC dextramer and anti-CD3 stained cells are determined. The frequency of specific MHC dextramer binding cells, or antigen specific T-cells towards TB specific peptide is calculated as “# of MHC dextramer positive, CD3+, CD8+ cells/total # of CD3+,CD8 positive cells”.
  • Process 37: Detection of TB Specific T Cells in Human Blood Using MHC Dextramers
  • A (sampling): Use of vacutainer or other container containing anti-coagulant to collect 4-10 ml human blood.
  • B (sample preparation): 10-500 μl of the full blood is transferred to a tube containing one or more MHC dextramers labeled with fluorochrome and carrying antigenic peptides derived from proteins from TB.
  • The MHC dextramers may all carry the same MHC I allele or carry different MHC I alleles.
  • During acute infection TB specific activated T cells will be present in increased amounts in an activated state compared to in healthy individuals. Also in individuals with latent TB specific T cells will be present. The presences of activated TB specific T cells may thereby act as a surrogate marker for active and/or latent infection with Mycobacterium tuberculosis. MHC multimers carrying TB specific peptides is in this example used to detect the presence of active and or latent TB specific T cells in the blood of patients infected with Mycobacterium tuberculosis.
  • Example TB proteins include but is not limited to Ag85b, Mtb39, CFP10, ESAT-6, Mtb8.4, Mtb9.9A, EsxG, any TB surface protein, any TB intracellular protein or any other TB protein from which peptide epitopes are presented by MHC I molecules.
  • The antigenic peptides are preferably 9′mers but can also be 8′mers, 10′ers or 11′mers.
  • The MHC dextramers are incubated for 1-60′ at room temperature in the dark, then gating reagents including labeled markers directed against CD3, CD8 and CD4 are added and incubation continued for 1-60′. RBC is lysed by addition of lysis reagents e.g. EasyLyse then the sample is centrifuged, supernatant removed.
  • Cells are washed by resuspension in buffer followed by centrifugation. Supernatant is removed and cells resuspended in buffer.
  • Optionally fixative is added.
  • C (assaying): The prepared sample is applied to a flow cytometer and data acquired.
  • D (data analysis): The obtained data are analyzed for the presences of cells stained with MHC dextramer, anti-CD3, anti-CD8 and not stained with anti-CD4.
  • E (interpretation of data): The presence of cells stained with MHC dextramer, anti-CD3, anti-CD8 and not stained with anti-CD4 are T cells specific for TB antigens and their frequency can optionally be determined as follows. The number of anti-CD8 and anti-CD3 stained cells and that of anti-CD8, MHC dextramer and anti-CD3 stained cells are determined. The frequency of specific MHC dextramer binding cells, or antigen specific T-cells towards TB specific peptide is calculated as “# of MHC dextramer positive, CD3+, CD8+ cells/total # of CD3+, CD8 positive cells”. Process 38: Detection of TB Specific T Cells in Human Blood Using a Lyse No-Wash Method.
  • A (sampling): Use of vacutainer or other container containing anti-coagulant to collect 4-10 ml human blood.
  • B (sample preparation): 10-500 μl of the full blood is transferred to a tube containing one or more MHC dextramers labeled with fluorochrome and carrying antigenic peptides derived from proteins from TB.
  • The MHC dextramers may all carry the same MHC I allele or carry different MHC I alleles.
  • During acute infection TB specific activated T cells will be present in increased amounts in an activated state compared to in healthy individuals. Also in individuals with latent TB specific T cells will be present. The presences of activated TB specific T cells may thereby act as a surrogate marker for active and/or latent infection with Mycobacterium tuberculosis. MHC multimers carrying TB specific peptides is in this example used to detect the presence of active and or latent TB specific T cells in the blood of patients infected with Mycobacterium tuberculosis.
  • Example TB proteins include but is not limited to Ag85b, Mtb39, CFP10, ESAT-6, Mtb8.4, Mtb9.9A, EsxG, any TB surface protein, any TB intracellular protein or any other TB protein from which peptide epitopes are presented by MHC I molecules.
  • The antigenic peptides are preferably 9′mers but can also be 8′mers, 10′ers or 11′mers.
  • The MHC dextramers are incubated for 1-30′ at room temperature in the dark, then gating reagents including labeled markers directed against CD3, CD8 and CD4 are added and incubation continued for 1-60′. RBC is lysed by addition of lysis reagents e.g. EasyLyse and buffer is added to dilute sample 2-50 times.
  • Optionally fixative is added.
  • C (assaying): The prepared sample is applied to a flow cytometer and data acquired.
  • D (data analysis): The obtained data are analyzed for the presences of cells stained with MHC dextramer, anti-CD3, anti-CD8 and not stained with anti-CD4.
  • E (interpretation of data): The presence of cells stained with MHC dextramer, anti-CD3, anti-CD8 and not stained with anti-CD4 are T cells specific for TB antigens and their frequency can optionally be determined as follows. The number of anti-CD8 and anti-CD3 stained cells and that of anti-CD8, MHC dextramer and anti-CD3 stained cells are determined. The frequency of specific MHC dextramer binding cells, or antigen specific
  • T-cells towards TB specific peptide is calculated as “# of MHC dextramer positive, CD3+, CD8+ cells/total # of CD3+, CD8 positive cells”.
  • Process 39: Detection of TB Specific T Cells in Sample of Purified Lymphocytes Derived from Human Blood
  • A (sampling): Use of vacutainer or other container containing anti-coagulant and cell separation medium to collect 4-10 ml human blood.
  • B (sample preparation): Vacutainer is centrifuged thereby separating cells according to size and lymphocytes are transferred to a new container. A fraction or all purified lymphocytes are added one or more MHC dextramers labeled with fluorochrome and carrying antigenic peptides derived from proteins from TB. The MHC dextramers may all carry the same MHC I allele or carry different MHC I alleles.
  • During acute infection TB specific activated T cells will be present in increased amounts in an activated state compared to in healthy individuals. Also in individuals with latent TB specific T cells will be present. The presences of activated TB specific T cells may thereby act as a surrogate marker for active and/or latent infection with Mycobacterium tuberculosis. MHC multimers carrying TB specific peptides is in this example used to detect the presence of active and or latent TB specific T cells in the blood of patients infected with Mycobacterium tuberculosis.
  • Example TB proteins include but is not limited to Ag85b, Mtb39, CFP10, ESAT-6, Mtb8.4, Mtb9.9A, EsxG, any TB surface protein, any TB intracellular protein or any other TB protein from which peptide epitopes are presented by MHC I molecules.
  • The antigenic peptides are preferably 9′mers but can also be 8′mers, 10′ers or 11′mers.
  • The sample and MHC dextramers are incubated for 1-30′ at room temperature in the dark, then gating reagents including labeled markers directed against CD3, CD8 and CD4 are added and incubation continued for 1-60′. The sample is centrifuged and supernatant removed. Cells are washed by resuspension in buffer followed by centrifugation. Supernatant is removed and cells resuspended in buffer.
  • Optionally fixative is added.
  • C (assaying): The prepared sample is applied to a flow cytometer and data acquired.
  • D (data analysis): The obtained data are analyzed for the presences of cells stained with MHC dextramer, anti-CD3, anti-CD8 and not stained with anti-CD4.
  • E (interpretation of data): The presence of cells stained with MHC dextramer, anti-CD3, anti-CD8 and not stained with anti-CD4 are T cells specific for TB antigens and their frequency can optionally be determined as follows. The number of anti-CD8 and anti-CD3 stained cells and that of anti-CD8, MHC dextramer and anti-CD3 stained cells are determined. The frequency of specific MHC dextramer binding cells, or antigen specific T-cells towards TB specific peptide is calculated as “# of MHC dextramer positive, CD3+, CD8+ cells/total # of CD3+, CD8 positive cells”.
  • Process 40: Detection of TB Specific T Cells in Sample of Human Blood Using a No-Lyse-No Wash Method.
  • A (sampling): Use of vacutainer or other container containing anti-coagulant to collect 4-10 ml human blood.
  • B (sample preparation): The blood is transferred to tube containing cell separation medium, e.g. ficoll. The tube is centrifuged thereby separating cells according to size and lymphocytes are transferred to a new container. A fraction or all purified lymphocytes are added one or more MHC dextramers labeled with fluorochrome and carrying antigenic peptides derived from proteins from TB.
  • During acute infection TB specific activated T cells will be present in increased amounts in an activated state compared to in healthy individuals. Also in individuals with latent TB specific T cells will be present. The presences of activated TB specific T cells may thereby act as a surrogate marker for active and/or latent infection with Mycobacterium tuberculosis. MHC multimers carrying TB specific peptides is in this example used to detect the presence of active and or latent TB specific T cells in the blood of patients infected with Mycobacterium tuberculosis.
  • Example TB proteins include but is not limited to Ag85b, Mtb39, CFP10, ESAT-6, Mtb8.4, Mtb9.9A, EsxG, any TB surface protein, any TB intracellular protein or any other TB protein from which peptide epitopes are presented by MHC I molecules.
  • The antigenic peptides are preferably 9′mers but can also be 8′mers, 10′ers or 11′mers.
  • The sample and MHC dextramers are incubated for 1-30′ at room temperature in the dark, then gating reagents including labeled markers directed against CD3, CD8 and
  • CD4 are added and incubation continued for 1-60′.
  • Buffer is added to dilute sample 2-50 times.
  • Optionally the sample is added fixative.
  • C (assaying): The prepared sample is applied to a flow cytometer and data acquired.
  • D (data analysis): The obtained data are analyzed for the presences of cells stained with MHC dextramer, anti-CD3, anti-CD8 and not stained with anti-CD4.
  • E (interpretation of data): The presence of cells stained with MHC dextramer, anti-CD3, anti-CD8 and not stained with anti-CD4 are T cells specific for TB antigens and their frequency can optionally be determined as follows. The number of anti-CD8 and anti-CD3 stained cells and that of anti-CD8, MHC dextramer and anti-CD3 stained cells are determined. The frequency of specific MHC dextramer binding cells, or antigen specific T-cells towards TB specific peptide is calculated as “# of MHC dextramer positive, CD3+, CD8+ cells/total # of CD3+, CD8 positive cells”.
  • Process 41: Detection of TB Specific T Cells in Blood in a No-Lyse No-Wash Assay.
  • A (sampling): Use of vacutainer or other container containing anti-coagulant to collect 4-10 ml human blood.
  • B (sample preparation): 10-500 μl blood is transferred to a tube and added one or more MHC dextramers labeled with fluorochrome and carrying antigenic peptides derived from proteins from TB.
  • Example TB proteins include but is not limited to Matrix phosphoprotein 65 (pp65), DNA polymerase, immediate early protein 1 (IE1), immediate early protein 3 (IE3), any intracellular protein, any surface protein or any other protein from which peptide epitopes are presented by MHC I molecules.
  • The antigenic peptides are preferably 9′mers but can also be 8′mers, 10′ers or 11′mers. During acute infection TB specific activated T cells will be present in increased amounts in an activated state compared to in healthy individuals. Also in individuals with latent TB specific T cells will be present. The presences of activated TB specific T cells may thereby act as a surrogate marker for active and/or latent infection with Mycobacterium tuberculosis. MHC multimers carrying TB specific peptides is in this example used to detect the presence of active and or latent TB specific T cells in the blood of patients infected with Mycobacterium tuberculosis.
  • Example TB proteins include but is not limited to Ag85b, Mtb39, CFP10, ESAT-6, Mtb8.4, Mtb9.9A, EsxG, any TB surface protein, any TB intracellular protein or any other TB protein from which peptide epitopes are presented by MHC I molecules.
  • The antigenic peptides are preferably 9′mers but can also be 8′mers, 10′ers or 11′mers.
  • The sample and MHC dextramers are incubated for 1-30′ at room temperature in the dark, then gating reagents including labeled markers directed against CD3, CD8, CD4, CD14 and CD45 are added and incubation continued for 1-60′. Preferably markers are labeled with label different from MHC dextramer label and different from each other.
  • Anti-CD4 and anti-CD14 markers are “dump” gate markers and can be labeled with same fluorochrome. Other dump gate markers like anti-CD19, anti-CD15 may also be included in analysis and are then labeled with same label as anti-CD4/anti-CD14 and added to the sample together with these markers.
  • Buffer is added to dilute sample 2-50 times.
  • Optionally, an exact volume and known number of beads (counting beads), e.g. CytoCount beads can be added to the sample.
  • Optionally the sample is added fixative.
  • C (assaying): The prepared sample is applied to a flow cytometer and data acquired.
  • D (data analysis): The obtained data are analyzed for the presences of cells stained with MHC dextramer, anti-CD3, anti-CD8 as follows. Cells stained positive with anti-CD4 and anti-CD14 are deselected in a “dump” gate and remaining cells stained positive with anti-CD45 are selected for further analysis. In this population the presences of cells stained with MHC dextramer, anti-CD3 and anti-CD8 markers are determined.
  • The amount of acquired beads is also determined.
  • E (interpretation of data): The presence of cells stained with MHC dextramer, anti-CD3 and anti-CD8 are T cells specific for TB antigens and their frequency can optionally be determined as follows. The number of anti-CD8 and anti-CD3 stained cells and that of anti-CD8, MHC dextramer and anti-CD3 stained cells are determined. The frequency of specific MHC dextramer binding cells, or antigen specific T-cells towards TB specific peptide is calculated as “# of MHC dextramer positive, CD3+, CD8+ cells/total # of CD3+, CD8 positive cells”.
  • If counting beads have been added the concentration of TB specific T cells can be determined more accurately using the following calculations:

  • Concentration of TB specific T cells=Dilution factor of blood×(# counted specific T cells×concentration of bead)/# counted beads.
  • Process 42: Detection of TB Specific T cells in Blood in a No-Lyse No-Wash Assay Using a Tube Containing Solidified Reagents.
  • A (sampling): Use of vacutainer or other tube containing anti-coagulant to collect 4-10 ml human blood.
  • B (sample preparation): 10-500 μl blood is transferred to a tube containing a solid or semisolid matrix featuring one or more MHC dextramers labeled with fluorochrome and carrying antigenic peptides derived from proteins from TB bacteria.
  • During acute infection TB specific activated T cells will be present in increased amounts in an activated state compared to in healthy individuals. Also in individuals with latent TB specific T cells will be present. The presences of activated TB specific T cells may thereby act as a surrogate marker for active and/or latent infection with Mycobacterium tuberculosis. MHC multimers carrying TB specific peptides is in this example used to detect the presence of active and or latent TB specific T cells in the blood of patients infected with Mycobacterium tuberculosis.
  • Example TB proteins include but is not limited to Ag85b, Mtb39, CFP10, ESAT-6, Mtb8.4, Mtb9.9A, EsxG, any TB surface protein, any TB intracellular protein or any other TB protein from which peptide epitopes are presented by MHC I molecules.
  • The antigenic peptides are preferably 9′mers but can also be 8′mers, 10′ers or 11′mers.
  • The matrix also features gating reagents including labeled markers directed against CD3, CD8, CD4, CD14 and CD45. Preferably markers are labeled with label different from MHC dextramer label and different from each other. Anti-CD4 and anti-CD14 markers are “dump” gate markers and can be labeled with same fluorochrome. Other dump-gate markers like anti-CD19, anti-CD15 or others may also be included in analysis and are then labeled with same label as anti-CD4/anti-CD14 markers and are also contained in matrix material.
  • The matrix material is such that MHC dextramers and gating reagents are released upon addition of blood. Preferably the matrix material is dissolved thereby releasing reagents into added sample.
  • The sample is incubated for 1-60′ at room temperature in the dark. Optionally additional gating reagents can be added. Buffer is added to dilute sample 2-50 times.
  • Optionally, an exact volume and known number of beads (counting beads), e.g. CytoCount beads can be added to the sample.
  • Optionally the sample is added fixative.
  • C (assaying): The prepared sample is applied to a flow cytometer and data acquired.
  • D (data analysis): The obtained data are analyzed for the presences of cells stained with MHC dextramer, anti-CD3, anti-CD8 as follows. Cells stained positive with anti-CD4 and anti-CD14 are deselected in a “dump” gate and remaining cells stained positive with anti-CD45 are selected for further analysis. In this population the presences of cells stained with MHC dextramer, anti-CD3 and anti-CD8 markers are determined.
  • The amount of acquired beads is also determined.
  • E (interpretation of data): The presence of cells stained with MHC dextramer, anti-CD3 and anti-CD8 are T cells specific for TB antigens and their frequency can optionally be determined as follows. The number of anti-CD8 and anti-CD3 stained cells and that of anti-CD8, MHC dextramer and anti-CD3 stained cells are determined. The frequency of specific MHC dextramer binding cells, or antigen specific T-cells towards TB specific peptide is calculated as “# of MHC dextramer positive, CD3+, CD8+ cells/total # of CD3+, CD8 positive cells”.
  • If counting beads have been added the concentration of TB specific T cells can be determined more accurately using the following calculations:

  • Concentration of TB specific T cells=Dilution factor of blood×(# counted specific T cells×concentration of bead)/# counted beads.
  • Process 43: Determination of absolute count of TB specific T cells in human blood using a two-panel technique.
  • A (sampling): Use of vacutainer or other tube containing anti-coagulant to collect 4-10 ml human blood.
  • B (sample preparation): 10-500 μl blood is transferred to a two tubes. The first tube is added anti-CD3, anti-CD4 and anti-CD8 markers labeled with different fluorochromes either before or after addition of blood.
  • The second tube are added anti-CD3, anti-CD4 and anti-CD8 markers labeled with different fluorochromes and also contains one or more MHC dextramers labeled with fluorochrome and carrying antigenic peptides derived from proteins from TB bacteria. The label of the individual MHC dextramers are either the same or different from each other. However all MHC dextramers are labeled with label(s) different for the labels used for the other marker molecules.
  • During acute infection TB specific activated T cells will be present in increased amounts in an activated state compared to in healthy individuals. Also in individuals with latent TB specific T cells will be present. The presences of activated TB specific T cells may thereby act as a surrogate marker for active and/or latent infection with Mycobacterium tuberculosis. MHC multimers carrying TB specific peptides is in this example used to detect the presence of active and or latent TB specific T cells in the blood of patients infected with Mycobacterium tuberculosis.
  • Example TB proteins include but is not limited to Ag85b, Mtb39, CFP10, ESAT-6, Mtb8.4, Mtb9.9A, EsxG, any TB surface protein, any TB intracellular protein or any other TB protein from which peptide epitopes are presented by MHC I molecules. The antigenic peptides are preferably 9′ mers but can also be 8′ mers, 10′ mers or 11′ mers.
  • Both tubes are incubated for 1-60′ at room temperature in the dark and lysis reagent is added, e.g. EasyLyse or iTAG MHC Tetramer Lyse reagent. The lysis reagent may optionally contain fixative.
  • Following incubation tube one is added buffer to dilute sample 0-50 times and an exact volume and known number of beads (counting beads), e.g. Flow-Count fluorospheres are added to tube one.
  • Tube two is centrifuged, supernatant removed and buffer added to wash cells. Following centrifugation supernatant is removed and cells resuspended in buffer with or without fixative.
  • C (assaying): The prepared samples are applied to a flow cytometer and data acquired.
  • D (data analysis): The obtained data for tube 1 are analyzed for the presences of cells stained with anti-CD3 and anti-CD8 markers and cells stained with anti-CD3 and anti-CD4 markers. The presence and amount of beads acquired is also analyzed. The obtained data for tube 2 are analyzed for the presence of cells stained with anti-CD3 and anti-CD8 markers, cells stained with anti-CD3 and anti-CD4 markers and also cells triple stained with MHC dextramers, anti-CD3 and anti-CD8 markers.
  • E (interpretation of data): The number of anti-CD8+, anti-CD3 double positive cells and the number of anti-CD4+, anti-CD3+ double positive cells acquired in tube one and tube 2 are determined. The number of acquired beads in tube one is also determined. The absolute concentration of CD3+, CD8+ positive cells can be calculated as follows:

  • Concentration of CD3+, CD8+ positive cells=(# counted CD3+, CD8+ positive cells×concentration of bead)/# counted beads.
  • Likewise the absolute concentration of CD3+, CD4+ positive cells can be calculated as follows: Concentration of CD3+, CD4+ positive cells pr. volume blood=(# counted CD3+, CD4+ positive cells×concentration of bead)/# counted beads.
  • The frequency of specific MHC dextramer binding cells, or antigen specific T-cells towards TB specific peptide is calculated as: (# of MHC dextramer positive, CD3+ and CD8+ cells)/(total # of CD3+, CD8+ positive cells).
  • Absolute concentration of TB specific T cells can then be calculated as follows:

  • (Absolute concentration of CD3+, CD8+ double positive cells)×(frequency of MHC dextramer+, CD3+, CD8+ triple positive cells).
  • Process 44: Determination of presences of TB specific T cells in human blood including a negative control reagent.
  • A (sampling): Use of vacutainer or other container containing anti-coagulant to collect 4-10 ml human blood.
  • B (sample preparation): 10-500 μl of the full blood is transferred to one or more tubes containing one or more MHC dextramers labeled with fluorochrome and carrying antigenic peptides derived from proteins from TB. MHC dextramers are MHC-peptide complexes e.g. generated by in vitro refolding, coupled to dextran through covalent or non-covalent interactions. The dextramer furthermore comprises fluorochrome coupled to the same dextran molecules through covalent or non-covalent interactions.
  • The MHC dextramers may all carry the same MHC I allele or carry different MHC I alleles.
  • During acute infection TB specific activated T cells will be present in increased amounts in an activated state compared to in healthy individuals. Also in individuals with latent TB specific T cells will be present. The presences of activated TB specific T cells may thereby act as a surrogate marker for active and/or latent infection with Mycobacterium tuberculosis. MHC multimers carrying TB specific peptides is in this example used to detect the presence of active and or latent TB specific T cells in the blood of patients infected with Mycobacterium tuberculosis.
  • Example TB proteins include but is not limited to Ag85b, Mtb39, CFP10, ESAT-6, Mtb8.4, Mtb9.9A, EsxG, any TB surface protein, any TB intracellular protein or any other TB protein from which peptide epitopes are presented by MHC I molecules. The antigenic peptides are preferably 9′ mers but can also be 8′ mers, 10′ mers or 11′ mers.
  • To another tube same volume of blood is added together with one or more MHC dextramer(s) labeled with fluorochrome and each carrying a negative control peptide. Negative control peptides are peptides binding the MHC allele of choice, but that does not support binding of the resultant MHC-peptide complex to the desired TCR. Example negative control peptides include but are not limited to modified peptides, naturally occurring peptide different from the peptide used for analysis and nonsense peptides (peptides with an amino acid sequence different from the linear sequence of any peptide derived from any known protein).
  • The antigenic peptides and/or negative control peptides are preferably 9′ mers but can also be 8′ mers, 10′ mers or 11′ mers.
  • All samples are incubated for 1-60′ at room temperature in the dark, then gating reagents including labeled markers directed against CD3, CD8 and CD4 are added and incubation continued for 1-60′. RBC is lysed by addition of lysis reagents e.g. EasyLyse then the sample is centrifuged, supernatant removed.
  • Cells are washed by resuspension in buffer followed by centrifugation. Supernatant is removed and cells resuspended in buffer.
  • Optionally fixative is added.
  • C (assaying): The prepared samples are applied to a flow cytometer and data acquired.
  • D (data analysis): The obtained data are analyzed for the presences of cells stained with MHC dextramer, anti-CD3, anti-CD8 and not stained with anti-CD4.
  • E (interpretation of data): Data from samples stained with MHC dextramers carrying negative control peptides are used as negative controls to assess the level of background fluorescence and non-specific binding of all MHC dextramers.
  • The presence of cells triple stained with MHC dextramers containing TB peptides, anti-CD3, anti-CD8 and not stained with anti-CD4 are T cells specific for TB antigens and their frequency can optionally be determined as follows. The number of anti-CD8 and anti-CD3 stained cells and that of anti-CD8, MHC dextramer and anti-CD3 stained cells are determined. The frequency of specific MHC dextramer binding cells, or antigen specific T-cells towards TB specific peptide is calculated as “# of MHC dextramer positive, CD3+ and CD8+ triple positive cells/total # of CD3+,CD8+ double positive cells”.
  • Negative control reagents comprising MHC dextramers carrying negative control peptides can be included in all processes involving staining with MHC dextramers.
  • Process 45: Detection of TB specific T cells in blood using MHC tetramers.
  • TB specific T cells may be detected and enumerated using MHC tetramers carrying TB specific peptides. MHC tetramers are Streptavidin or Avidin associated with 4 biotinylated MHC-peptide molecules and labeled with flourochrome e.g. APC or PE.
  • Preferred processes involving use of MHC tetramers carrying TB specific peptides for detection of TB specific T cells include processes similar to process 36-44 but where MHC tetramers are used in stead of MHC dextramers.
  • Process 46: Detection of TB specific T cells in blood using MHC multimers.
  • TB specific T cells may be detected and enumerated using MHC multimers carrying TB specific peptides. MHC multimers are complexes comprising of more than one MHC-peptide complexes, held together by covalent or non-covalent interactions between a multimerization domain and one or more MHC-peptide complexes and where the multimerizations domain is a molecule, a complex of molecules, a cell or a solid support.
  • Preferred processes involving use of MHC multimers carrying TB specific peptides for detection of TB specific T cells include processes similar to process 36-44 but where MHC multimers are used instead of MHC dextramers.
  • Process 47: Detection of cancer specific T cells in human blood using MHC dextramers A (sampling): Use of vacutainer or other container containing anti-coagulant to collect 4-10 ml human blood.
  • B (sample preparation): 10-500 μl of the full blood is transferred to a tube containing one or more MHC dextramers labeled with fluorochrome and carrying antigenic peptides derived from one or more proteins over expressed in cancer cells, also called cancer antigens. Example cancer antigen include but is not limited to gp100, MAGE-1, MAGE-3, Her-2, MART-1, p53, survivin, telomerase, Bcl-X, Bcl-2, NyESO, Prostate Specific Antigen (PSA), Tyrosinase, HPV E6/E7, hTERT, MUC1, CEA, Melan-A, WT1, EGFRvIII or any other cancer related antigen.
  • The antigenic peptides are preferably 9′ mers but can also be 8′ mers, 10′ mers or 11′ mers.
  • The MHC dextramers are incubated for 1-60′ at room temperature in the dark, then gating reagents including labeled markers directed against CD3, CD8 and CD4 are added and incubation continued for 1-60′. RBC is lysed by addition of lysis reagents e.g. EasyLyse then the sample is centrifuged, supernatant removed.
  • Cells are washed by resuspension in buffer followed by centrifugation. Supernatant is removed and cells resuspended in buffer.
  • Optionally fixative is added.
  • C (analysis): The prepared sample is applied to a flow cytometer and data acquired.
  • D (data analysis): The obtained data are analyzed for the presences of cells stained with MHC dextramer, anti-CD3, anti-CD8 and not stained with anti-CD4.
  • E (interpretation of data): The presence of cells stained with MHC dextramer, anti-CD3, anti-CD8 and not stained with anti-CD4 are T cells specific for cancer antigens and their frequency can optionally be determined as follows. The number of anti-CD8 and anti-CD3 stained cells and that of anti-CD8, MHC dextramer and anti-CD3 stained cells are determined. The frequency of specific MHC dextramer binding cells, or antigen specific T-cells towards Cancer antigen specific peptide is calculated as “# of MHC dextramer positive, CD3+, CD8+ cells/total # of CD3+, CD8 positive cells”.
  • Process 48: Detection of Cancer specific T cells in human blood using a lyse no-wash method.
  • A (sampling): Use of vacutainer or other container containing anti-coagulant to collect 4-10 ml human blood.
  • B (sample preparation): 10-500 μl of the full blood is transferred to a tube containing one or more MHC dextramers labeled with fluorochrome and carrying antigenic peptides derived from one or more proteins over expressed in cancer cells, also called cancer antigens. Example cancer antigen include but is not limited to gp100, MAGE-1, MAGE-3, Her-2, MART-1, p53, survivin, telomerase, Bcl-X, Bcl-2, NyESO, Prostate Specific Antigen (PSA), Tyrosinase, HPV E6/E7, hTERT, MUC1, CEA, Melan-A, WT1, EGFRvIII or any other cancer related antigen.
  • The antigenic peptides are preferably 9′ mers but can also be 8′ mers, 10′ mers or 11′ mers.
  • The MHC dextramers are incubated for 1-30′ at room temperature in the dark, then gating reagents including labeled markers directed against CD3, CD8 and CD4 are added and incubation continued for 1-60′. RBC is lysed by addition of lysis reagents e.g. EasyLyse and buffer is added to dilute sample 2-50 times.
  • Optionally fixative is added.
  • C (analysis): The prepared sample is applied to a flow cytometer and data acquired.
  • D (data analysis): The obtained data are analyzed for the presences of cells stained with MHC dextramer, anti-CD3, anti-CD8 and not stained with anti-CD4.
  • E (interpretation of data): The presence of cells stained with MHC dextramer, anti-CD3, anti-CD8 and not stained with anti-CD4 are T cells specific for Cancer antigens and their frequency can optionally be determined as follows. The number of anti-CD8 and anti-CD3 stained cells and that of anti-CD8, MHC dextramer and anti-CD3 stained cells are determined. The frequency of specific MHC dextramer binding cells, or antigen specific T-cells towards Cancer specific peptide is calculated as “# of MHC dextramer positive, CD3+, CD8+ cells/total # of CD3+, CD8 positive cells”.
  • Process 49: Detection of Cancer specific T cells in sample of purified lymphocytes derived from human blood
  • A (sampling): Use of vacutainer or other container containing anti-coagulant and cell separation medium to collect 4-10 ml human blood.
  • B (sample preparation): Vacutainer is centrifuged thereby separating cells according to size and lymphocytes are transferred to a new container. A fraction or all purified lymphocytes are added one or more MHC dextramers labeled with fluorochrome and carrying antigenic peptides derived from one or more proteins over expressed in cancer cells, also called cancer antigens. Example cancer antigen include but is not limited to gp100, MAGE-1, MAGE-3, Her-2, MART-1, p53, survivin, telomerase, Bcl-X, Bcl-2, NyESO, Prostate Specific Antigen (PSA), Tyrosinase, HPV E6/E7, hTERT, MUC1, CEA, Melan-A, WT1, EGFRvIII or any other cancer related antigen.
  • The antigenic peptides are preferably 9′ mers but can also be 8′ mers, 10′ mers or 11′ mers.
  • The sample and MHC dextramers are incubated for 1-30′ at room temperature in the dark, then gating reagents including labeled markers directed against CD3, CD8 and CD4 are added and incubation continued for 1-60′. The sample is centrifuged and supernatant removed. Cells are washed by resuspension in buffer followed by centrifugation. Supernatant is removed and cells resuspended in buffer.
  • Optionally fixative is added.
  • C (analysis): The prepared sample is applied to a flow cytometer and data acquired.
  • D (data analysis): The obtained data are analyzed for the presences of cells stained with MHC dextramer, anti-CD3, anti-CD8 and not stained with anti-CD4.
  • E (interpretation of data): The presence of cells stained with MHC dextramer, anti-CD3, anti-CD8 and not stained with anti-CD4 are T cells specific for Cancer antigens and their frequency can optionally be determined as follows. The number of anti-CD8 and anti-CD3 stained cells and that of anti-CD8, MHC dextramer and anti-CD3 stained cells are determined. The frequency of specific MHC dextramer binding cells, or antigen specific T-cells towards Cancer specific peptide is calculated as “# of MHC dextramer positive, CD3+, CD8+ cells/total # of CD3+, CD8 positive cells”.
  • Process 50: Detection of cancer specific T cells in sample of human blood using a no-lyse-no wash method.
  • A (sampling): Use of vacutainer or other container containing anti-coagulant to collect 4-10 ml human blood.
  • B (sample preparation): The blood is transferred to tube containing cell separation medium, e.g. ficoll. The tube is centrifuged thereby separating cells according to size and lymphocytes are transferred to a new container. A fraction or all purified lymphocytes are added one or more MHC dextramers labeled with fluorochrome and carrying antigenic peptides derived from one or more proteins over expressed in cancer cells, also called cancer antigens. Example cancer antigen include but is not limited to gp100, MAGE-1, MAGE-3, Her-2, MART-1, p53, survivin, telomerase, Bcl-X, Bcl-2, NyESO, Prostate Specific Antigen (PSA), Tyrosinase, HPV E6/E7, hTERT, MUC1, CEA, Melan-A, WT1, EGFRvIII or any other cancer related antigen.
  • The antigenic peptides are preferably 9′ mers but can also be 8′ mers, 10′ mers or 11′ mers.
  • The sample and MHC dextramers are incubated for 1-30′ at room temperature in the dark, then gating reagents including labeled markers directed against CD3, CD8 and CD4 are added and incubation continued for 1-60′.
  • Buffer is added to dilute sample 2-50 times.
  • Optionally the sample is added fixative.
  • C (analysis): The prepared sample is applied to a flow cytometer and data acquired.
  • D (data analysis): The obtained data are analyzed for the presences of cells stained with MHC dextramer, anti-CD3, anti-CD8 and not stained with anti-CD4.
  • E (interpretation of data): The presence of cells stained with MHC dextramer, anti-CD3, anti-CD8 and not stained with anti-CD4 are T cells specific for Cancer antigens and their frequency can optionally be determined as follows. The number of anti-CD8 and anti-CD3 stained cells and that of anti-CD8, MHC dextramer and anti-CD3 stained cells are determined. The frequency of specific MHC dextramer binding cells, or antigen specific T-cells towards Cancer specific peptide is calculated as “# of MHC dextramer positive, CD3+, CD8+ cells/total # of CD3+, CD8 positive cells”.
  • Process 51: Detection of cancer specific T cells in blood in a no-lyse no-wash assay. A (sampling): Use of vacutainer or other container containing anti-coagulant to collect 4-10 ml human blood.
  • B (sample preparation): 10-500 μl blood is transferred to a tube and added one or more MHC dextramers labeled with fluorochrome and carrying antigenic peptides derived from one or more proteins over expressed in cancer cells, also called cancer antigens. Example cancer antigen include but is not limited to gp100, MAGE-1, MAGE-3, Her-2, MART-1, p53, survivin, telomerase, Bcl-X, Bcl-2, NyESO, Prostate Specific Antigen (PSA), Tyrosinase, HPV E6/E7, hTERT, MUC1, CEA, Melan-A, WT1, EGFRvIII or any other cancer related antigen.
  • The antigenic peptides are preferably 9′ mers but can also be 8′ mers, 10′ mers or 11′ mers.
  • The sample and MHC dextramers are incubated for 1-30′ at room temperature in the dark, then gating reagents including labeled markers directed against CD3, CD8, CD4, CD14 and CD45 are added and incubation continued for 1-60′. Preferably markers are labeled with label different from MHC dextramer label and different from each other. Anti-CD4 and anti-CD14 markers are “dump” gate markers and can be labeled with same fluorochrome. Other dump gate markers like anti-CD19, anti-CD15 may also be included in analysis and are then labeled with same label as anti-CD4/anti-CD14 and added to the sample together with these markers.
  • Buffer is added to dilute sample 2-50 times.
  • Optionally, an exact volume and known number of beads (counting beads), e.g. CytoCount beads can be added to the sample.
  • Optionally the sample is added fixative.
  • C (analysis): The prepared sample is applied to a flow cytometer and data acquired.
  • D (data analysis): The obtained data are analyzed for the presences of cells stained with MHC dextramer, anti-CD3, anti-CD8 as follows. Cells stained positive with anti-CD4 and anti-CD14 are deselected in a “dump” gate and remaining cells stained positive with anti-CD45 are selected for further analysis. In this population the presences of cells stained with MHC dextramer, anti-CD3 and anti-CD8 markers are determined.
  • The amount of acquired beads is also determined.
  • E (interpretation of data): The presence of cells stained with MHC dextramer, anti-CD3 and anti-CD8 are T cells specific for Cancer antigens and their frequency can optionally be determined as follows. The number of anti-CD8 and anti-CD3 stained cells and that of anti-CD8, MHC dextramer and anti-CD3 stained cells are determined. The frequency of specific MHC dextramer binding cells, or antigen specific T-cells towards Cancer specific peptide is calculated as “ # of MHC dextramer positive, CD3+, CD8+ cells/total # of CD3+, CD8 positive cells”.
  • If counting beads have been added the concentration of Cancer specific T cells can be determined more accurately using the following calculations:

  • Concentration of Cancer specific T cells=Dilution factor of blood×(# counted specific T cells×concentration of bead)/# counted beads.
  • Process 52: Determination of absolute count of cancer specific T cells in human blood using a two-panel technique.
  • A (sampling): Use of vacutainer or other tube containing anti-coagulant to collect 4-10 ml human blood.
  • B (sample preparation): 10-500 μl blood is transferred to a two tubes. The first tube is added anti-CD3, anti-CD4 and anti-CD8 markers labeled with different fluorochromes either before or after addition of blood.
  • The second tube are added anti-CD3, anti-CD4 and anti-CD8 markers labeled with different fluorochromes and also contains one or more MHC dextramers labeled with fluorochrome and carrying antigenic peptides derived from proteins over expressed in cancer cells. The label of the individual MHC dextramers are either the same or different from each other. However all MHC dextramers are labeled with label(s) different for the labels used for the other marker molecules.
  • Example cancer antigen include but is not limited to gp100, MAGE-1, MAGE-3, Her-2, MART-1, p53, survivin, telomerase, Bcl-X, Bcl-2, NyESO, Prostate Specific Antigen (PSA), Tyrosinase, HPV E6/E7, hTERT, MUC1, CEA, Melan-A, WT1, EGFRvIII or any other cancer related antigen.
  • The antigenic peptides are preferably 9′ mers but can also be 8′ mers, 10′ mers or 11′ mers.
  • Both tubes are incubated for 1-60′ at room temperature in the dark and lysis reagent is added, e.g. EasyLyse or iTAG MHC Tetramer Lyse reagent. The lysis reagent may optionally contain fixative.
  • Following incubation tube one is added buffer to dilute sample 0-50 times and an exact volume and known number of beads (counting beads), e.g. Flow-Count fluorospheres are added to tube one.
  • Tube two is centrifuged, supernatant removed and buffer added to wash cells.
  • Following centrifugation supernatant is removed and cells resuspended in buffer with or without fixative.
  • C (analysis): The prepared samples are applied to a flow cytometer and data acquired.
  • D (data analysis): The obtained data for tube 1 are analyzed for the presences of cells stained with anti-CD3 and anti-CD8 markers and cells stained with anti-CD3 and anti-CD4 markers. The presence and amount of beads acquired is also analyzed.
  • The obtained data for tube 2 are analyzed for the presence of cells stained with anti-CD3 and anti-CD8 markers, cells stained with anti-CD3 and anti-CD4 markers and also cells triple stained with MHC dextramers, anti-CD3 and anti-CD8 markers.
  • E (interpretation of data): The number of anti-CD8+, anti-CD3 double positive cells and the number of anti-CD4+, anti-CD3+ double positive cells acquired in tube one and tube 2 are determined. The number of acquired beads in tube one is also determined. The absolute concentration of CD3+, CD8+ positive cells can be calculated as follows:

  • Concentration of CD3+, CD8+ positive cells =(# counted CD3+, CD8+ positive cells×concentration of bead)/# counted beads.
  • Likewise the absolute concentration of CD3+, CD4+ positive cells can be calculated as follows: Concentration of CD3+, CD4+ positive cells pr. volume blood =(# counted CD3+, CD4+ positive cells×concentration of bead)/# counted beads.
  • The frequency of specific MHC dextramer binding cells, or antigen specific T-cells towards Cancer specific peptide is calculated as: (# of MHC dextramer positive, CD3+and CD8+ cells)/(total # of CD3+, CD8+ positive cells).
  • Absolute concentration of Cancer specific T cells can then be calculated as follows:

  • (Absolute concentration of CD3+, CD8+ double positive cells)×(frequency of MHC dextramer+, CD3+, CD8+ triple positive cells).
  • Process 53: Determination of presences of cancer specific T cells in human blood including a negative control reagent.
  • A (sampling): Use of vacutainer or other container containing anti-coagulant to collect 4-10 ml human blood.
  • B (sample preparation): 10-500 μl of the full blood is transferred to one or more tubes containing one or more MHC dextramers labeled with fluorochrome and carrying antigenic peptides derived from one or more proteins over expressed in cancer cells, also called cancer antigens. Example cancer antigen include but is not limited to gp100, MAGE-1, MAGE-3, Her-2, MART-1, p53, survivin, telomerase, Bcl-X, Bcl-2, NyESO, Prostate Specific Antigen (PSA), Tyrosinase, HPV E6/E7, hTERT, MUC1, CEA, Melan-A, WT1, EGFRvIII or any other cancer related antigen.
  • The MHC dextramers may all carry the same MHC I allele or carry different MHC I alleles.
  • To another tube same volume of blood is added together with one or more MHC dextramer(s) labeled with fluorochrome and each carrying a negative control peptide.
  • Negative control peptides are peptides binding the MHC allele of choice, but that does not support binding of the resultant MHC-peptide complex to the desired TCR. Example negative control peptides include but are not limited to modified peptides, naturally occurring peptide different from the peptide used for analysis and nonsense peptides (peptides with an amino acid sequence different from the linear sequence of any peptide derived from any known protein).
  • The antigenic peptides and/or negative control peptides are preferably 9′ mers but can also be 8′ mers, 10′ mers or 11′ mers.
  • All samples are incubated for 1-60′ at room temperature in the dark, then gating reagents including labeled markers directed against CD3, CD8 and CD4 are added and incubation continued for 1-60′. RBC is lysed by addition of lysis reagents e.g. EasyLyse then the sample is centrifuged, supernatant removed.
  • Cells are washed by resuspension in buffer followed by centrifugation. Supernatant is removed and cells resuspended in buffer.
  • Optionally fixative is added.
  • C (analysis): The prepared samples are applied to a flow cytometer and data acquired.
  • D (data analysis): The obtained data are analyzed for the presences of cells stained with MHC dextramer, anti-CD3, anti-CD8 and not stained with anti-CD4.
  • E (interpretation of data): Data from samples stained with MHC dextramers carrying negative control peptides are used as negative controls to assess the level of background fluorescence and non-specific binding of all MHC dextramers. The presence of cells triple stained with MHC dextramers containing cancer peptides, anti-CD3, anti-CD8 and not stained with anti-CD4 are T cells specific for Cancer antigens and their frequency can optionally be determined as follows. The number of anti-CD8 and anti-CD3 stained cells and that of anti-CD8, MHC dextramer and anti-CD3 stained cells are determined. The frequency of specific MHC dextramer binding cells, or antigen specific T-cells towards Cancer specific peptide is calculated as “# of MHC dextramer positive, CD3+ and CD8+ triple positive cells/total # of CD3+,CD8+double positive cells”.
  • Negative control reagents comprising MHC dextramers carrying negative control peptides can be included in all processes involving staining with MHC dextramers.
  • Process 54: Detection of cancer specific T cells in blood using MHC tetramers. Cancer specific T cells may be detected and enumerated using MHC tetramers carrying Cancer specific peptides. MHC tetramers are Streptavidin or Avidin associated with 4 biotinylated MHC-peptide molecules and labeled with flourochrome e.g. APC or PE.
  • Preferred processes involving use of MHC tetramers carrying Cancer specific peptides for detection of Cancer specific T cells include processes similar to process 7-13, 15-22 but where MHC tetramers are used in stead of MHC dextramers.
  • Process 55: Detection of cancer specific T cells in blood using MHC multimers. Cancer specific T cells may be detected and enumerated using MHC multimers carrying Cancer specific peptides. MHC multimers are complexes comprising of more than one MHC-peptide complexes, held together by covalent or non-covalent interactions between a multimerization domain and one or more MHC-peptide complexes and where the multimerizations domain is a molecule, a complex of molecules, a cell or a solid support.
  • Preferred processes involving use of MHC multimers carrying Cancer specific peptides for detection of Cancer specific T cells include processes similar to process 7-13, 15-22 but where MHC multimers are used instead of MHC dextramers.
  • Process 56: Detection of specific CD8+ T cells in human blood.
  • A (sampling): Use of any container as described elsewhere herein containing anti-coagulant to collect 4-10 ml human blood.
  • B (sample preparation): A fraction of the blood sample is transferred to a tube together with one or more MHC I dextramers or other MHC I multimers labeled with fluorochrome and carrying antigenic peptides. The MHC dextramers/multimers may all carry the same MHC allele or each carry a different MHC alleles.
  • The antigenic peptides are preferably 9′ mers but can also be 8′ mers, 10′ mers or 11′ mers.
  • The MHC dextramers are incubated for 1-60′ at room temperature in the dark, then gating reagents including labelled anti-CD3, anti-CD8 markers are added and incubation continued for 1-60′. Optionally other gating reagents are added together with the anti-CD3 and anti-CD8 marker molecules. RBC is lysed by addition of lysis reagents e.g. EasyLyse then buffer is added to dilute sample 0-20 times and sample is ready for analysis.
  • Alternatively the sample is centrifuged, supernatant removed. Cells are washed by resuspension in buffer followed by centrifugation. Washing step may be repeated. Supernatant is removed and cells resuspended in buffer.
  • Optionally fixative is added either together with lysis reagent or following washing of cells.
  • Optionally, an exact volume and known number of beads (counting beads), e.g. CytoCount beads can be added to the sample prior to analysis.
  • C (assaying): The prepared sample is applied to a flow cytometer and data acquired.
  • D (data analysis): The obtained data are analyzed for the presences of cells triple stained with MHC dextramer/multimer, anti-CD3 and anti-CD8.
  • If counting beads have been added to the sample, the amount of acquired beads is also determined.
  • E (interpretation of data): The presence of cells triple stained with MHC dextramer, anti-CD3, anti-CD8 are T cells specific for the antigen(s) binding the peptide binding cleft of the MHC molecules of the MHC dextramer/multimer reagents and their frequency can y be determined as follows. The number of anti-CD8 and anti-CD3 double stained cells and that of anti-CD8, MHC dextramer and anti-CD3 triple stained cells are determined. The frequency of specific MHC dextramer binding cells, or antigen specific T-cells towards Borrelia specific peptide is calculated as “# of MHC dextramer positive, CD3+, CD8+ cells/total # of CD3+,CD8 positive cells”.
  • If counting beads have been added the concentration of antigen specific T cells can be determined more accurately using the following calculations:

  • Concentration of antigen specific T cells=Dilution factor of blood×(# counted specific T cells×concentration of bead)/# counted beads.
  • Process 57: Detection of specific CD4+ T cells in human blood.
  • A (sampling): Use of any container as described elsewhere herein containing anti-coagulant to collect 4-10 ml human blood.
  • B (sample preparation): A fraction of the blood sample is transferred to a tube together with one or more MHC II dextramers or other MHC II multimers labeled with fluorochrome and carrying antigenic peptides. The MHC dextramers/multimers may all carry the same MHC allele or each carry a different MHC alleles.
  • The antigenic peptides are preferably 12-18′ mers but can also be 9′ mers, 10′ mers, 11′ mers, 19′ mers, 20′ mers or even longer peptide fragments.
  • The MHC dextramers are incubated for 1-60′ at room temperature in the dark, then gating reagents including labelled anti-CD3, anti-CD4 markers are added and incubation continued for 1-60′. Optionally other gating reagents are added together with the anti-CD3 and anti-CD8 marker molecules. RBC is lysed by addition of lysis reagents e.g. EasyLyse then buffer is added to dilute sample 0-20 times and sample is ready for analysis.
  • Alternatively the sample is centrifuged, supernatant removed. Cells are washed by resuspension in buffer followed by centrifugation. Washing step may be repeated. Supernatant is removed and cells resuspended in buffer.
  • Optionally fixative is added either together with lysis reagent or following washing of cells.
  • Optionally, an exact volume and known number of beads (counting beads), e.g. CytoCount beads can be added to the sample prior to analysis.
  • C (assaying): The prepared sample is applied to a flow cytometer and data acquired.
  • D (data analysis): The obtained data are analyzed for the presences of cells triple stained with MHC dextramer/multimer, anti-CD3 and anti-CD4.
  • If counting beads have been added to the sample, the amount of acquired beads is also determined.
  • E (interpretation of data): The presence of cells triple stained with MHC dextramer, anti-CD3, anti-CD4 are T cells specific for the antigen(s) binding the peptide binding cleft of the MHC molecules of the MHC dextramer/multimer reagents and their frequency can y be determined as follows. The number of anti-CD4 and anti-CD3 double stained cells and that of anti-CD4, MHC dextramer and anti-CD3 triple stained cells are determined. The frequency of specific MHC dextramer binding cells, or antigen specific T-cells towards Borrelia specific peptide is calculated as “# of MHC dextramer positive, CD3+, CD4+ cells/total # of CD3+,CD4 positive cells”.
  • Process 58: Detection of specific CD8+ T cells in sample of purified lymphocytes derived from human blood
  • A (sampling): Use of vacutainer or other container containing anti-coagulant and cell separation medium to collect 4-10 ml human blood.
  • B (sample preparation): Vacutainer is centrifuged thereby separating cells according to size and lymphocytes are transferred to a new container. A fraction or all purified lymphocytes are added one or more MHC dextramers labeled with fluorochrome and carrying antigenic peptides derived from any antigenic protein.
  • The MHC dextramers/multimers may all carry the same MHC allele or each carry a different MHC alleles. The MHC molecules of the present process are mHC I molecules.
  • The antigenic peptides are preferably 9′ mers but can also be 8′ mers, 10′ mers or 11′ mers.
  • The sample and MHC dextramers are incubated for 1-30′ at room temperature in the dark, then gating reagents including labeled markers directed against CD3, CD8 and CD4 are added and incubation continued for 1-60′. The sample is centrifuged and supernatant removed. Cells are washed by resuspension in buffer followed by centrifugation. Supernatant is removed and cells resuspended in buffer.
  • Optionally fixative is added.
  • C (analysis): The prepared sample is applied to a flow cytometer and data acquired.
  • D (data analysis): The obtained data are analyzed for the presences of cells stained with MHC dextramer, anti-CD3, anti-CD8 and not stained with anti-CD4.
  • E (interpretation of data): The presence of cells stained with MHC dextramer, anti-CD3, anti-CD8 and not stained with anti-CD4 are T cells specific for specific antigens and their frequency can optionally be determined as follows. The number of anti-CD8 and anti-CD3 stained cells and that of anti-CD8, MHC dextramer and anti-CD3 stained cells are determined. The frequency of specific MHC dextramer binding cells, or antigen specific T-cells towards specific antigenic peptide is calculated as “# of MHC dextramer positive, CD3+, CD8+ cells/total # of CD3+, CD8 positive cells”.
  • Process 59: Detection of specific CD4+ T cells in sample of purified lymphocytes derived from human blood
  • A (sampling): Use of vacutainer or other container containing anti-coagulant and cell separation medium to collect 4-10 ml human blood.
  • B (sample preparation): Vacutainer is centrifuged thereby separating cells according to size and lymphocytes are transferred to a new container. A fraction or all purified lymphocytes are added one or more MHC dextramers labeled with fluorochrome and carrying antigenic peptides derived from any antigenic protein.
  • The MHC dextramers/multimers may all carry the same MHC allele or each carry a different MHC alleles. The MHC molecules of the present process are MHC II molecules.
  • The antigenic peptides are preferably 12-18′ mers but can also be 9′ mers, 10′ mers, 11′ mers, 19′ mers, 20′ mers or even longer peptide fragments.
  • The sample and MHC dextramers are incubated for 1-30′ at room temperature in the dark, then gating reagents including labeled markers directed against CD3, CD8 and CD4 are added and incubation continued for 1-60′. The sample is centrifuged and supernatant removed. Cells are washed by resuspension in buffer followed by centrifugation. Supernatant is removed and cells resuspended in buffer.
  • Optionally fixative is added.
  • C (analysis): The prepared sample is applied to a flow cytometer and data acquired.
  • D (data analysis): The obtained data are analyzed for the presences of cells stained with MHC dextramer, anti-CD3, anti-CD4 and not stained with anti-CD8.
  • E (interpretation of data): The presence of cells stained with MHC dextramer, anti-CD3, anti-CD4 and not stained with anti-CD8 are T cells specific for specific antigens and their frequency can optionally be determined as follows. The number of anti-CD4 and anti-CD3 stained cells and that of anti-CD4, MHC dextramer and anti-CD3 stained cells are determined. The frequency of specific MHC dextramer binding cells, or antigen specific T-cells towards specific antigenic peptide is calculated as “# of MHC dextramer positive, CD3+, CD4+ cells/total # of CD3+, CD4 positive cells”.
  • Process 60: Determination of antigen specific T cells in human blood using measurement of secretion of cytokine captured on solid support
  • A (sampling): Use any container as described elsewhere herein containing anticoagulant, e.g. a vacutainer to collect blood.
  • B (sample preparation): The blood is transferred to a new tube containing cell separation medium, e.g. ficoll. Optionally the blood is diluted in buffer prior to addition to tube. The tube is centrifuged thereby separating cells according to size and the peripheral blood lymphocyte (PBMC) layer are transferred to a new container and washed with buffer and/or cell culturing medium. A fraction of the purified PBMC's is frozen using a standard cell freezing protocol know by persons skilled in the art. The remaining PBMC's are plated in wells of cell culture plates, antigenic peptides added and incubated for 1-5 days at 37° C., 5% CO2. Then T cell stimulating interleukine, e.g. IL-2 is added and incubation continued for 1-4 days.
  • Frozen PBMC's are thawed, washed and irradiated and then added to the culture wells together with more antigenic peptides. Incubation is continued for another day.
  • C (Assaying):
  • The prepared PBMC's from the wells described above are transferred to an equal number of wells of microtiter plates with immobilized INF-γ at the bottom of each well (capture plates). The capture plates are incubated for 37° C. for 12-24 hours then cells discharged allowing INF-γ secreted by the added cells to bind immobilized anti-INF-γ antibody. Then plates are washed and enzyme-labeled antibody or other marker molecule able to bind INF-γ is added to each well and the plate incubated for 30-240 minutes at room temperature. The anti-INF-γ marker molecule may be labeled itself or alternatively detected through a secondary enzyme-labeled marker molecule. Following wash substrate for the enzyme is added and the plate incubated to allow conversion of substrate into measurable product and then the reaction stopped by e.g. washing. Examples of useful enzyme-substrate pairs are described elsewhere herein.
  • Optionally the plates are dried e.g. over night.
  • Images of spots corresponding to the INF-y secreted by individual antigen-stimulated T cells are captured with a video camera and the images analyzed by appropriate software e.g. NIH image software or other commercially available or customized software applications able to count the spots.
  • Alternatively, spots can be quantitated manually by a technician using standard light microscopy. Spots can also be counted manually under a light microscope.
  • D (data analysis): Captured images are enhanced using the Look Up Table which contrasts the images. Thresholding is then applied to every image and a wand tool is used to highlight the border to effectively subtract the edge of the well so that background counts won't be high and artificial. Density slicing over a narrow range is then used to highlight the spots produced from secreting cells. Pixel limits are set to subtract out small debris and large particles, and the number of spots falling within the prescribed pixel range are counted by the software program. Totals from each well are then manually recorded for future analysis.
  • E (interpretation of data): Each counted spot represents INF-y secreted by one antigen-stimulated T cells. The presence of spots therefore correlates to the presence of antigen specific T cells in the original sample.
  • The amounts of T cells in the originally sample can be described as the concentration of T cells in sample=(# counted spots×dilution of blood sample)/volume of sample assayed
  • Process 61: Determination of TB specific T cells in human blood using measurement of secretion of cytokine captured on solid support
  • A (sampling): Use any container as described elsewhere herein containing anticoagulant, e.g. a vacutainer to collect blood.
  • B (sample preparation): The blood is transferred to a new tube containing cell separation medium, e.g. ficoll. Optionally the blood is diluted in buffer prior to addition to tube. The tube is centrifuged thereby separating cells according to size and the peripheral blood lymphocyte (PBMC) layer are transferred to a new container and washed with buffer and/or cell culturing medium. A fraction of the purified PBMC's is frozen using a standard cell freezing protocol know by persons skilled in the art. The remaining PBMC's are plated in wells of cell culture plates, antigenic peptides derived from TB proteins are added and incubated for 1-5 days at 37° C., 5% CO2. Then T cell stimulating interleukine, e.g. IL-2 is added and incubation continued for 1-4 days. Frozen PBMC's are thawed, washed and irradiated and then added to the culture wells together with more TB specific antigenic peptides. Incubation is continued for another day.
  • C (Assaying):
  • The prepared PBMC's from the wells described above are transferred to an equal number of wells of microtiter plates with immobilized INF-γ at the bottom of each well (capture plates). The capture plates are incubated for 37° C. for 12-24 hours then cells discharged allowing INF-γ secreted by the added cells to bind immobilized anti-INF-γ antibody. Then plates are washed and enzyme-labeled antibody or other marker molecule able to bind INF-γ is added to each well and the plate incubated for 30-240 minutes at room temperature. The anti-INF-γ marker molecule may be labeled itself or alternatively detected through a secondary enzyme-labeled marker molecule. Following wash substrate for the enzyme is added and the plate incubated to allow conversion of substrate into measurable product and then the reaction stopped by e.g. washing. Examples of useful enzyme-substrate pairs are described elsewhere herein.
  • Optionally the plates are dried e.g. over night.
  • Images of spots corresponding to the INF-γ secreted by individual antigen-stimulated T cells are captured with a video camera and the images analyzed by appropriate software e.g. NIH image software or other commercially available or customized software applications able to count the spots.
  • Alternatively, spots can be quantitated manually by a technician using standard light microscopy. Spots can also be counted manually under a light microscope.
  • D (data analysis): Captured images are enhanced using the Look Up Table which contrasts the images. Thresholding is then applied to every image and a wand tool is used to highlight the border to effectively subtract the edge of the well so that background counts won't be high and artificial. Density slicing over a narrow range is then used to highlight the spots produced from secreting cells. Pixel limits are set to subtract out small debris and large particles, and the number of spots falling within the prescribed pixel range are counted by the software program. Totals from each well are then manually recorded for future analysis.
  • E (interpretation of data): Each counted spot represents INF-γ secreted by one TB antigen-stimulated T cells. The presence of spots therefore correlates to the presence of TB specific T cells in the original sample.
  • The amounts of TB specific T cells in the originally sample can be described as the concentration of T cells in sample =(# counted spots x dilution of blood sample)/volume of sample assayed
  • Process 62: Detection of cancer specific marker in solid tissue
  • A (sampling): Tissue, fixed and embedded in paraffin are sliced in thin slices and placed on a glass slice.
  • B (sample preparation): The paraffin block is sliced and thin slices are placed on glass slices. The sample is deparafinated, followed by a antigen retrival, and stained for DNA using DAPI and subsequent incubation with detection reagents, substrate is added and allowed to react.
  • C (assaying): The markers used are; a antibody specific for cancer specific marker on the surface of cell or inside cell. The antibody is coupled to HRP reagent (Horse Radish Peroxidase coubled), and DAPI stain is used for cell identification. The presence of cells expressing the cancer marker is detected by the precipitation of a dye due to the HRP enzyme.
  • D (Data acquistion): The sample is analyzed manually by light microscopy, or by digital imaging.
  • E (Interpretation of Data): The amount of cells expressing the cancer specific marker are determined by the precipitated color, correlated to the DAPI stain for positive cell identification, either manually of by automated software working on the digital image.
  • Process 63: Phenotyping a sample using a lyse-wash procedure and flow cytometry
  • A (sampling): Use of vacutainer to collelt 5 ml human blood.
  • B (sample preparation): 10-100 μl of blood is transferred to a tube together with fluorochrome labeled antibodies specific for marker molecules on cells in sample. The sample is incubated at room temperature for 1-60′ in the dark. Lysis reagent e.g. EasyLyse or another lysis reagent as described elsewhere herein is added to lyse RBC. Optionally fixative is added to the sample together with lysis reagent. Sample is centrifuged, supernatant removed and cells resuspended in buffer. For compensation controls the same procedure are performed on individual cell samples with markers labeled with each of the flurochromes used on individual cell samples. E.g. if three markers are used with three different fluorochromes three individual samples are prepared with markers labeled with the respective flurochromes. The samples for compensation controls can be identical to or different from the analysed sample. C (assaying): The prepared sample is applied to a flow cytometer and data acquired. D (data analysis): The obtained flow cytometry data are analysed, first the spectral overlap are compensated for, using automatic compensation software, on data from compensation control sample i.e. single stained samples.
  • The presence of cells stained with the individual marker molecules are then determined. Optionally cells labeled with more than one marker are identified. E (interpretation of data): The number of cells labeled with the different marker is determined. The percentage of individual subpopulations of cells are calculated.
    • Examples Example 1 “Matrix Protocol for MHC Class I Specific T-cell Quantization Performed on Human Blood”
  • In this example the following sub-processes are used:
      • Sampling: Blood retrieval from person using a vacutainer, containing an anticoagulation agent.
      • Sample preparation: Fructose/Trehalose (matrix) and fluorescent markers were present in the assay container in a Matrix. No-lyse protocol was followed, i.e. the red blood cells were not lysed.
      • Assaying: The analysis involves the use of the following marker molecules: anti-CD45 antibody (binds to the CD45 membrane protein of leucocytes), anti-CD3 antibody (binds to CD3 protein on surface of T cells), anti-CD8 antibody (binds to CD8 receptor on surface of cytotoxic T cells and NK cells), and MHCCMVDex, binds to HLD protein on the surface on antigen specific cytotoxic T cells. CytoCOunt™ beads, for quantitation of positive cell/volume sample.
      • Acquisition: Data was acquired using the Summit™ software of the CyAn ADP™ flow cytometer
      • Sorting:ND
      • Interpretation of data: Data were analyzed using the Summit™ software, and Quantitation of the concentration of antigen specific Cytotoxic T Cells
      • Manipulation of entities: ND
  • Human Blood has been collected using a 5 ml vacutainer containing EDTA as anticoagulants. Exactly 50.0 μl full blood was transferred to a “matrix”—tube. The “matrix” contained anti-CD45/Fluor1, anti-CD3 Fluor2, anti-CD8/Fluor3, MHCCMVDex/Fluor4 reagents, and 105 counting beads. In this case anti-CD45/CY, anti-CD3/PB, anti-CD8/Alexa700, MHCCMVDex/RPE and the CytoCount™ beads has been used. MHCHIVDex/RPE for which the donor is negative was used as negative control. Preparation of the “Matrix” tubes; briefly, solutions of 20% Fructose in water and 20% Trehalose in water were made and mixed in a 1:1 ratio. 15 μl of this mixture were transferred to 5 ml Falcon tubes. A premix of antibodies were made consisting of 40 μl anti-CD8/Alexa700 labeled antibody in a concentration of 25 μg/ml+′μl anti-CD3/Pasific Blue labeled antibody in a concentration of 100 μg/ml+160 μl anti-CD45/Cascade Yellow labeled antibody in a concentration of 200 μg/ml. 12 μl of this mixture were added to each Falcon tube. 100 μl butylated hydroxytoluen (BHT) with a concentration of 99 mg/L were added. The mixtures were dried under vacuum a 2-8° C. over night.
  • MHCCMVDEX/RPE is HLA-B *0702 loaded with the peptide (TPRVTTGGGAM), the MHCHIVDex/RPE is HLA-B *0702 loaded with the peptide (TPGPGVRYRL). After mixing the “Matrix” reagents and the blood together, the mix was incubated for 15 min at room temperature, and subsequently acquired on a CyAn ADP™ flow cytometer equipped with a 488, 405 and a 638 nm laser.
  • The instrument PMT was adjusted using anti-CD45/CY stained sample, otherwise unstained samples, and the trigger parameter used was anti-CD45/CY antibody signal. Compensation controls; the fluorochrome spillover coefficients for the experiment was determined by running “single” stain controls, either as “Matrix” stubs or normal lyse and washed samples. As the samples had to be acquired using a fluorochromes signal as trigger parameter we used the method of co-staining the “single” color controls with anti-CD45/Fluor, which signal is used as trigger parameter, which has no spillover into the stain for which it was used as stain control; thus for RPE, PB and CY spillover determination, was used anti-CD3/Fluor co-stain with anti-CD45/APC for triggering, for APC and A700 spillover determination was used anti-CD3/Flour costained with anti-CD45/PB. Summit™ software was used to determine the spillover coefficients and subsequently the compensation matrix was used during the analysis of the data in Summit™ flowcytometry software.
  • Analysis and Gating Strategy:
  • The goal was to identify the MHCCMV class I specific binding T-Cells in peripheral blood from a specific individual. As a No-lyse procedure was used, gating on scatter parameters can not be done properly, thus anti-CD45/CY vs Side scatter (SSC) was used for initial gating. HLA-Class I specific cells are a sub population of CD8 positive T-Cells, therefore anti-CD3/PB vs. anti-CD8/Alexa700 was used as a second gating criteria (FIG. 2). Subsequently, the anti-CD3 positive population was analyzed in a plot showing anti-CD8 and MHCCMVDex positive events, in this plot the double positive events were identified (FIG. 2B)
  • Enumeration of MHCCMVDex positive cells (FIG. 2B). Based on the negative control, the count obtained in the last gate described in “FIG. 2Bc” were subtracted for unspecific binding and background events identified in the negative control sample (FIG. 2A). Using the quantitative counting beads, CytoCount™ as internal standard, the concentration of MHCCMVDex specific T-cells was defined:

  • (Count of MHCCMV +×Bead concentration/Bead count in CMV)−(Count of MHCHIV×Bead concentration/Bead count in HIV); where the bead count is the count of bead in the respective sample (FIG. 2):

  • [(351×2×106bead/ml)/22584Bead]−[(97×2×106bead/ml)/21929Bead]=31084Cell/ml−8846 Cell/ml=22238 MHCCMV class I specific T-Cells/ml full blood
    • Example 2 “A Lyse and Wash (LW) Protocol has been Employed for Chemical Contra Selection of the RBC”
  • In this example the following sub-processes are used:
      • Sampling: Primary Vacutainer®, containing EDTA as anticoagulant Sample
      • Preparation: Lyse and wash, with light fixation, using Utilyse™
      • Assaying: Markers was identical to example 1, but here using Scatter parameters as primary gate criteria.
      • Acquisition: Data was acquired using the Summit™ software of the CyAn ADP™ flow cytometer
      • Sorting: ND
      • Interpretation of data: Data were analyzed using the Summit™ software, Quantitation of the concentration of antigen specific Cytotoxic T Cells
      • Manipulation of entities: ND
  • The sample, 100 μl peripheral human blood was added 100 μl UtiLyse™ solution A (Dako Cat. No. S3350), for a light sample fixation. After 10 min. incubation 2 ml UtiLyse™ solution B, the lysing reagents containing ammonium, were added and the sample were incubated 10 min at room temperature. Subsequently, the sample was centrifuged 300g for 5 min, the supernatant was discharge and the cell-pellet was resuspended in 500 μl of Phosphate buffer saline pH7.2. The cell sample was added into the “Matrix tubes as described in example 1, stained and analyzed accordingly on a CyAn ADP™ flow cytometer. By lysing the RBC, it enables use of the scatter parameters for gating FIG. 3, in contrast to NL (No lysis of RBC), where a trigger reagent (in this case anti-CD45/CY) was required for gating.
  • The flow Cytometry data was analyzed in the same way as shown in example 1, except that the initial gating was performed using the FSC and SSC parameters, gating on the lymphocyte population of cells, as seen in FIG. 3. The rest of the phenotyping and quantitation was identical to that of example 1.
    • Example 3 “Physical Selected Mononucleated Cells and Subsequent Analyzed Using Flowcytometre”
  • In this example the following sub-processes are used:
      • Sampling: Peripheral blood form a human being, in a open blood glass.
      • Sample preparation: Mononucleated cell fractioning using ficoll density centrifugation.
      • Assaying: The analysis involves the use of the following marker molecules: anti-CD3 antibody (binds to the CD3 T cell receptor), anti-CD8 antibody (binds to CD8 receptor on surface of cytotoxic T cells and NK cells), and MHCCMVDex, binds to HLD protein on the surface on antigen specific cytotoxic T cells.
      • CytoCount™ was added in the sample for enumeration of a antigen specific T-Cell population.
      • Acquisition: Data was acquired using the Summit™ software of the CyAn ADP™ flow cytometer.
      • Sorting: ND.
      • Interpretation of data: Data were analyzed using the Summit™ software, and Quantitation of the concentration of antigen specific Cytotoxic T Cells.
      • Manipulation of entities: ND
  • Human Perical blood was subjected to a ficoll density centrifugation, and the mononucleated cell fraction was subjected to immunophenotyping, and specific T-Cell enumeration using the gating antibodies anti-CD3/RPE, anti-CD8/PB. For specific enumeration of the CMV specific T-Cells, APC labeled HLA-A0101MHC-Dex molecule complexed with the CMV peptide (VTEHDTLLY) was used. A negative control sample was used, labeled with the same antibody markers, but with a specific APC labeled HLA-A0101MHC-Dex molecule complexed with the HIV peptide (ILKEPVHGV) for which the donor was negative. The phenotyping, analysis and enumeration were performed as in example 2, and example 1, respectively.
    • Example 4 “Phenotyping the Major Lymphocyte Sub Populations in Human peripheral Blood, Using Different Detections Molecules in Multicolor Flowcytometry”
  • In this example the following sub-processes are used:
      • Sampling: Human peripheral blood sampled in a vacutainer®.
      • Sample preparation: Lysis of RBC using Utilyse™.
      • Assaying: The analysis involves the use of the following marker molecules:
      • Direct labels/fluorochromes to antibody conjugate; anti-CD3/FITC, anti-CD56/RPE, anti-CD45/PB and anti-CD20/APC. Biotin conjugated antibodies; anti-CD5, anti-CD8, anti-CD19, anti-CD4, anti-CD14, anti-CD15, subsequent labeled using streptavidine Qdot®'s, each specific detection molecule was generated by mixing the biotinylated antibody with the respective Qdot®. A mix of the different detection molecules was made and used to phenotype the leucocytes.
      • This is also an example of the use of a dump gate using two antibodies with the same labeling molecule; anti-CD14-biotin and anti-CD15 biotin labeled with the same Qdot®.
      • Exemplify calculation of spillover signal of FITC detected in the RPE dedicated channel.
      • Acquisition: Data was acquired using the Summit™ software of the CyAn ADP™ flow cytometer.
      • Sorting: ND.
      • Interpretation of data: Data were analyzed using the Summit™ software, to phenotype this blood sample.
      • Manipulation of entities: ND
  • Sampling of human peripheral blood were performed using a vacutainer with EDTA as anticoagulant, followed by chemical elimination of RBC from human peripheral blood sample was done using Uti-Lyse™ fixing and lyseing reagent, as described in example 2.
  • The detection molecules used in this staining were direct fluorochrome conjugated antibodies; anti-CD3/FITC, anti-CD56/RPE, anti-CD45/PB and anti-CD20/APC, (Dako cat. no. F0818, R7251, PB0983). The antibodies; anti-CD5, anti-CD8, anti-CD19, anti-CD4, anti-CD14, anti-CD15 were all conjugated to Biotin, for subsequent labeling through the biotin streptavidine interaction with streptavidine coupled Qdot™ which are quantum clusters having the ability to emit light at a specific wavelength when exited by the lasers in the flow cytometer. The Biotin conjugates were premixed with strepavidine Qdots to give the following reagents; anti-CD8/Qdot655, anti-CD19/Qdot605, anti-CD4/Q705, (anti-CD14 + anti-CD15)/Qdot800. anti-CD5 was used as a premix reagent, bound to Goat anti mouse Qdot655 conjugate.
  • The amount of primary antibodies, Biotin conjugates and Qdot streptavidine reagent as well as the ratio between the binding pairs was optimized for the ability to stain positive cell populations, judged by the staining intensity and the unspecific association with the negative cell population for each antibody. Staining with the antibody biotin conjugate bound to the Qdots™ were performed using a reagent premix; each pair of antibody biotin conjugate and streptavidine Qdot reagents were mixed, followed by 15 min incubation. A 20 fold molar excess of free biotin, to the number of biotin binding sits on the streptavidine-Qdots™ reagents, was added to block all free biotin biding sits on the streptavidine. In this way reducing the likelihood of an unbound or detach antibody biotin molecule, from re-bind to another streptavidine Qdot™ during the incubation with the sample. The staining was performed by adding each antibody conjugates and antibody Qdot premix, into 50 μl of RBC depleated human blood, followed by a 15-30 min incubation at room temperature. The amount of reagents added to the premixes were; 1.5 pmol Qdot streptavidine/antibody premix, ˜2-6 pmol (˜0.3-1 μg) primary antibody biotin conjugate, or antibody fluorochrome conjugate, and approximately 20 fold molar excess of free biotin.
  • The staining were analyzed on a flow cytometer, having 3 laser lines 405, 488 and 635 nm for excitation of the fluorochromes and Qdot™ associated with the cell populations. The data files were stored on a computer accessible from the internet, anywhere in the world.
  • The gating strategy was to “purify” the lymphocyte population from any monocytes (anti-CD14 positive), and granulocytes (anti-CD15 positive), using the anti-CD14 and anti-CD15 antibodies both labeled with the same label, namely Qdot™ 800. This enabled contra selection of both granulocyts and monocyts occupying only one detector, and fluorochrome (FIG. 4). Subsequent the phenotyping was performed on the clean lymphocyte population FIG. 6.
  • Compensation for spectral overlaps are required in multicolor phenotyping, because of spectral overlap between many of the fluorochromes and Qdot™ associated with the same cell. Determination of the spillover coefficients were performed by single staining for each of the used detections markers, and detecting the signal in all the detectors involved. This is exemplified in, FIG. 5, for anti-CD3/FITC spillover into the detector dedicated RPE detection. The slope of the straight line observed when all event are plotted in a dot plop is the spillover coefficient, in this case calculated as the difference in median value of the Dim and High population in each channel, divided with each other, which is a better estimate for the real spillover than using the negative and the High population:
  • (RPEHigh-RPEDim)/(FITCHigh-FITCDim)=(225.39-91.37)/(101.82-39.81)=46,3%, which shows that 46,3% of the FITC signal will be detected in the RPE channel, with the settings for this particular experiment. By doing this manually or by the help of a software for each detector and fluorochrome used, mathematical linear algebra can by solved for each event/cells, and the real intensity for each fluorochrome in the dedicated detector can be found. In FIG. 5 and FIG. 6, the compensated intensities for the multicolor stains are shown. Each population of cells is characterized by the particular markers bound, and the intensity of the signal from each marker.
    • Example 5 “Quantification of a CD34 Positive Cells Population in a Sample where the DNA Binding Fluorescent Marker, 7-Aminoactinomycin D (7-AAD), was used for Counter Selection of Dead Cells by Flow Cytometry”
  • In this example the following sub-processes are used:
      • Sampling: Using a Vacutainer®.
      • Sample preparation: using a nonfixating ammonium based, EacyLyse™, the RBC were lysed.
      • Assaying: The analysis involves the use of the following marker molecules: anti-CD45 antibody (binds to the CD45 protein at the surface of leucocytes), anti-CD34 antibody (binds to surface CD34 molecule on hemapoetic stem cells),
      • Scatter parameters. CytoCount™ was added in the sample for enumeration of the CD34 positive cells.
      • Exemplify, staining of DNA by 7-AAD as a marker for dead cells in the sample.
      • Acquisition: Data was acquired using the Summit™ software of the CyAn ADP™ flow cytometer.
      • Sorting: ND.
      • Interpretation of data: Data were analyzed using the Summit™ software, and enumeration of the CD34 positive cells.
      • Manipulation of entities: ND
  • CD34 positive; is a rare cell type in peripheral blood. Antibody fluorochrome conjugates are used to identify the subpopulation in which to find the CD34 cells. 7-AAD is a fluorescent molecule that interact and bind to DNA, however the DNA is only accessible in cells that are dead or dying, thus these cells was labeled with 7-AAD, and counter selected for the phenotyping and counting of the CD34 cells. The actual concentration of CD34 cells are accessed using a fluorescent bead as counting standard (CytoCount™), in the same way as described in example 1, for enumeration of antigen specific T Cells.
  • Staining of sample and addition of count control beads
  • The sample was diluted to about 107 leucocytes per ml with PBS, counted by microscopy or by a cell counter. 100 μl of the physical purified leucocytes or peripheral blood are pipetted into a test tube. The sample was stained by addition of 10 μl anti-CD45/FITC and anti-CD34/RPE antibody conjugates, and mixed.
  • After incubation at room temperature for 20 min. the RBC were depleted form the sample using 2 ml of lysing reagent (EasyLyse ™ diluted 20×), and incubation for 10 min inn the dark.
  • Viability staining, 7-AAD staining of dead cells was performed by addition of 10 μl of 0.01% 7-AAD solution to each sample and subsequently mixed.
  • The standardized counting of CD34 cells in the sample was performed by addition of exactly 100 μl fluorescent counting beads (CytoCount™), e.i. 106 beads/sample. The sample was analyzed on a flow cytometer, by acquiring a minimum of 60 000 white blood cell, or 1000 CytoCount™ beads. Due to the high number of events, the file size become very high, an exclusions gate was placed in the area of the scatter picture known to include only lysed cells, RBC and debris, FIG. 7. In this way the number of event recorded in the data file, thus the size, of the file was dramatically reduced.
  • The first step of identifying the CD34 viable cells was counter selection of dead, 7-AAD stained cells, using a Side scatter vs. 7-AAD plot, FIG. 8. The defined viable cells are now phenotyped, analyzed for the antibody markers added, and the CD34 viable cells identified using a complicated gating strategy called ISHAGE, published by “Sutherland D R, Anderson L, Keeney M, Nayar R, Chin-Yee I. The ISHAGE guidelines for CD34positive cell determination by flow cytometry. J Hematother 1996;5:213-26.”
  • Compensation for spectral overlap between the used fluorescing markers were essential, and performed as single stains as described in example 4, however using the fluorochromes used in this example.
  • The results obtained is a specific concentration of viable CD34 cells, in this case only 66 cells/μl sample, or a frequency of CD34 positive cells of approximately 0.37% of the viable cells.
    • Example 6 “MHC-Dextramer Staining of Antigen Specific T-Cells on Frozen Tissue Sections Using Enzymatic Chromogenic Precipitation Detection”
  • In this example the following sub-processes are used:
      • Sampling: Tissue obtained form mouse spleen, frozen and sliced, and subsequent placed on a glass slice.
      • Sample preparation: Frozen tissue and preparation of cryosections, subsequent fixated by acetone.
      • Assaying: FITC labeled MHCCMVDex, binds to HLD protein on the surface of antigen specific cytotoxic T cells. Second layer detection using anti-FITC antibody conjugated to HRP
      • Acquisition: HRP mediated precipitation of colored precipitate, evaluated by light microscopy.
      • Sorting: ND.
      • Interpretation of data: ND.
      • Manipulation of entities: ND
  • Equilibrate the cryosection tissue (e.g. section of spleen from transgenic mice) to −20° C. in the cryostate. Cut 5 μm sections and then dry sections on slides at room temperature. Store slides frozen until use at −20° C.
  • Equilibrate frozen sections to room temperature. Fix with acetone for 5 min.
  • Immediately after fixation transfer slides to TBS buffer (50 mM Tris- HCL pH 7,6, 150 mM NaCl) for 10 min.
  • Incubate slides with FITC-conjugated MHC-dextramers at appropriate dilution (1:40-1:80) and incubate for 30 min at room temperature. Other dilution ranges, as well as incubation time and temperature, may be desirable.
  • Decant solution and gently tap slides against filter paper, submerge in TBS buffer.
  • Decant and wash for 10 min in TBS buffer.
  • Incubate with rabbit polyclonal anti-FITC antibody (Dako P5100) at 1:100 dilution in TBS at room temperature for 30 min.
  • Repeat step 5 and 6.
  • Incubate with Envision anti-Rabbit HRP (Dako K4003) at room temperature for 30 min.
  • Other visualization systems may be used.
  • Repeat step 5 and 6.
  • Develop with DAB+(Dako K3468) in fume hood for 10 min. Other substrates may be used
  • Rinse slides in tap-water for 5 min.
  • Counterstain with hematoxylin (Dako S3309) for 2 min.
  • Repeat step 12, mount slides.
  • The slides stained with MHC-Dextramers can now be evaluated by microscopy.
    • Example 7 “MHC-Dextramer Staining of Antigen Specific T-Cells on Frozen Tissue Sections Using Fluorescently Detection”
  • In this example the following sub-processes are used:
      • Sampling: Frozen tissue and preparation of cryosections.
      • Sample preparation: Acetone fixation.
      • Assaying: The analysis involves the use of the following marker molecules: anti-CD8 antibody labeled by a fluorochrome different from FITC (binds to CD8 receptor on surface of cytotoxic T cells and NK cells), and MHCCMVDex/FITC labeled, binds to HLD protein on the surface on antigen specific cytotoxic T cells.
      • Acquisition: Data was acquired using a fluorescent microscope enquired with two filters specific for FITC (for the MHC binding cells) and TRIC (for the CD8 binding cells), respectively.
      • A DNA label DAPI is also used for identification of cells in general.
      • Sorting: ND.
      • Interpretation of data: Cells (DAPI stained) that are costained for both MHCdex and CD8 molecule represent the antigen specific cytotoxic T cells.
      • Manipulation of entities: ND
  • In addition to MHC-dextramer staining it is desirable to co-stain with other antibodies of interest (e.g. anti-CD8). Fluorescently visualization can be used to evaluate both antibodies and MHC-dextramers on the same slide.
      • Acetone fixed frozen tissue section as described in the example above.
  • Incubate slides with anti-CD8. Use fluorescently labeled anti-CD8 (e.g. TRIC) or add a secondary fluorescently labeled antibody.
  • Wash slides.
  • Incubate slides with MHC-dextramers (e.g. FITC labeled dextramers).
  • Wash slides.
  • Mount slides (e.g. in mounting media containing DAPI).
  • The slides stained with antibodies and MHC-dextramers may now be evaluated in fluorescent microscopy. Overlay of image containing antibody (e.g. anti-CD8) and image containing MHC-dextramers can be used to identify cells fluorescently labeled with both reagents. This can be used to identify e.g. CD8 positive T-cells positive for a relevant MHC-dextramer.
    • Example 8 “MHC-Dextramer Staining of Antigen Specific T-Cells from Formaldehyde Fixed and Paraffin-Embedded Tissue Sections by Immunohistochemistry (IHC) and Digital Imaging”
  • In this example the following sub-processes are used:
      • Sampling: Tissue, formaldehyde fixed and paraffin-embedded tissue section.
      • Sample preparation: Deparafination, addition of markers and subsequent of secondary labeling layer, using HRP as label.
      • Assaying: FITC labeled MHCCMVDex, binds to HLD protein on the surface of antigen specific cytotoxic T cells. Second layer detection using anti-FITC antibody conjugated to HRP
      • Acquisition: HRP mediated precipitation of colored precipitate, and evaluation by light microscopy, and digital imaging.
      • Sorting: ND.
      • Interpretation of data: The sample is analyzed for the presence of the antigen specific cytotoxic T Cells, automatically or manually.
      • Manipulation of entities: ND
  • Formaldehyde fixed paraffin-embedded tissue are cut in section and mounted on the glass slice, for subsequent IHC staining with MHC-dextramers. Tissue fixed and prepared according to other protocols may be used as well. E.g. fresh tissue, lightly fixed tissue section (e.g. tissue fixed in 2% formaldehyde) or formalin-fixed, paraffin-embedded tissue section.
  • Optimal staining may require target retrieval treatment with enzymes as well as heating in a suitable buffer before incubation with antibodies and MHC-dextramer.
  • The sample is stained for DNA using DAPI stain, followed by incubated with an antigen specific MHCdex/FITC reagent, followed by addition of anti-FITC antibody labeled with HRP.
  • Then the substrate for HRP, “DAP” is added and the reaction allows to progress.
  • The sample is analyzed by light microscopy for the present of a colored precipitate on the cells (DAPI stained nucleus) positive for the specific MHC/dex reagent.
  • A digital image of the stained sample is obtained, and this can be analyzed manually in the same way as by microscopy. However, a digital image may be used for automatic determination of where and how many cells that are positive, related to the total amount of cells, determined by the DAPI staining, or other criteria or stainings.
    • Example 9 “MHC'mer Binding to TCRs Immobilized on Beads”
  • In this example the following sub-processes are used:
      • Sampling: Specific volume taken form the particle suspension
      • Sample preparation: Specific MHCdex were prepared, and mixed with the TCR coubled beads.
      • Assaying: TCRs immobilized on carboxylated polysturene beads, used to test the ability of a MHCdex preparation ability to binde the specific TCR.
      • Acquisition: Solid support quantitation, where target (specific TCR) is bound to a particle. Bound MHCdex/fluorochrome is detected by flowcytometry of the bead suspension.
      • Sorting: ND.
      • Interpretation of data: The binding ability of the specific MHCdex/fluorochrome to the TCR coupled beads is identified by staining of the beads with the fluorochrome carried by the specific MHCdex molecule.
      • Manipulation of entities: ND
  • This example describes how the quality of a MHC'mer may be tested, using the principles of solid support where target is bound to the particle. The MHC'mer is in this example a MHC-dextramer, and the test involves specific binding of the MHC-dextramer to TCRs immobilized on polysturene beads.
  • Recombinant TCRs (CMV3 TCRs; Soluble CMVpp65(NLVPMVATV)-specific TCR protein) specific for the MHC-peptide complex HLA-A*0201(NLVPMVATV), where the letters in parenthesis denote the peptide complexed to the MHC-allel HLA-A*0201, were obtained from Altor Biosciences. The TCRs were dimers linked via an IgG framework.
  • The purity of the TCRs was verified by SDS PAGE. The quality of the TCRs were verified by their ability to recognize the relevant MHC-dextramer and not irrelevant MHC dextramers in ELISA experiments (data not shown).
  • Carboxylate-modified beads were coupled with dimeric TCR (CMV3 TCRs; Soluble CMVpp65(NLVPMVATV)-specific TCR protein), incubated with fluorescently labeled MHC-dextramers and the extend of cell staining analysed by flow cytometry, as follows:
  • Immobilization of TCR on carboxylate beads:
  • 3×109 Carboxylate-modified beads, Duke Scientific Corporation, XPR-1536, 4 μm, lot:4394 were washed in 2×500 μl Wash buffer 1 (0,05% Tetronic 1307, 0,1M MES-buffer (2-[N-morpholino]ethanesulfonic acid), pH 6,0), centrifuged 4 min at 15000 g, and the supernatant was discarded.
  • 125 μl EDAC/Sulfo-NHS (50 mM EDAC (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide), 50 mM Sulfo-NHS, in Wash buffer 1) was added to the beads, and the suspension incubated at room temperature for 20 min.
  • Beads were washed in 2×250 μl Wash buffer 1 and centrifuged 2 min at 15000 g, and the supernatant was discarded.
  • TCR was added in various concentrations from 0 μg to 20 μg, and incubated with slow shaking overnight at 4° C.
  • Beads were centrifuged 4 min at 15000 g, and the supernatant discarded.
  • Beads were washed in 2×500 μl Wash buffer 1 and centrifuged 4 min at 1500 g, and the supernatant was discarded.
  • 125 μl 20 mM Glycin in Wash buffer 1 was added, and resuspended beads incubated for 1 hour at room temperature.
  • Beads were washed in 2×500 μl phosphate-buffered saline (PBS) pH 7.2, 0.5% Tetronic 1307, and centrifuged 2 min at 15000 g, and the supernatant was discarded.
  • Beads were resuspended in 250 μl PBS pH 7.2, 0.05% Tetronic 1307.
  • Bead concentration after resuspension was 1.2×107 beads/μl. Beads coated with TCR are stored at 2-8° C.
  • Several different protein concentrations and reaction buffers (e.g. acetate, phosphate or borate buffers) at different pH should be investigated to optimize the conjugation. Flow cytometry analysis:
  • 20 μl beads (1.2×107 beads/μl) coated with 0-20 μg TCRs, as described above. Beads were washed in 200 μl Wash buffer 2 (5% FCS, PBS, pH 7.4).
  • Beads were centrifuged 3 min at 12000 g, and the supernatant was discarded, and beads resuspended in 50 μl Wash buffer 2.
  • 10 μl MHC-dextramers were added, and samples were incubated 15 min. at room temperature in the dark.
  • Samples were washed in 1 ml Wash buffer 2, centrifuged at 300 g for 5 min. The supernatant was discarded, and pellet resuspended in 0,4 ml PBS pH 7.4, and kept at 4° C. in the dark until analysis on flow cytometer.
  • Samples were analysed by flow cytometry on a CyAn ADP™ instrument.
  • The specific results found were as listed in the following; MHC-Complex, TCR on Bead, and % positive events, respectively.
  •
    HLA-A*0201(NLVPMVATV) 20 μg dimeric TCR 99.8%
    HLA-A*0201(NLVPMVATV) 10 μg dimeric TCR 99.8%
    HLA-A*0201(NLVPMVATV)  5 μg dimeric TCR 99.7%
    HLA-A*0201(NLVPMVATV)  2 μg dimeric TCR 93.1%
    HLA-A*0201(NLVPMVATV)  0 μg dimeric TCR 25.1%
    HLA-A*0201(ILKEPVHGV) 10 ug dimeric TCR 1.5%
    • Example 10 “How to Used Only Two Different Fluorochromes, e.g. FITC and PB for Labeling of Multiple Antibodies, Simultaneously”
  • In this example the following sub-processes are used:
      • Sampling: Any process to get human blood sample.
      • Sample preparation: Lysis of RBC, or no lysis, the sample may or may not be fixed.
      • Assaying: Anti CD3, anti-CD4, anti-CD8 and anti-CD19, each stained with a specific ratio of the two fluorochromes FITC and PB, but may be any other pairs of fluorochromes with no spectral overlap in-between.
      • Acquisition: Is performed by flowcytometry capable of detecting the fluorochromes employed.
      • Sorting: The samples may be sorted by the markers used.
      • Interpretation of data: The data may be subjected to linear algebra calculation to deconvolute the marker related parameters from the FITC and PB staining measured.
      • Manipulation of entities: ND
  • In Multicolor flow Cytometry, the advantage is that it is possible to investigate the presence of many specific caresterics on a single entity, e.g. cell at the same time. However, there is a limit to that mount of fluorochromes that is accessible for the investigator, as well as many flow instruments only has few detectors.
  • This example is a description on how to used only two different fluorochromes, e.g. FITC and PB for labeling of multiple marker molecules, e.g. antibodies.
  • The principle is to label an antibody with a known specific ratio between the two fluorochromes, thus the intensity ration between the FITC and the PB detector will become a new parameter. In theory any ratio difference between the fluorochromes that can be measured will give raise to a new parameter.
  • In this example the samples have been prepped as in example 1. The characteristics that is analyze is the presence of 3 specific antibodies, such as anti-CD3, anti-CD4 and anti-CD8, anti-CD19. Each antibody is labeled with a combination of two fluorochromes, FITC (F) and PB (P). Labeling of the antibodies are performed by chemical conjugation to a defined mix of FITC and PB, or one of the two fluorochromes alone. In this way the amount of required detectors to use for 4 detections molecules is reduced to only two detectors on the flow cytometer. The antibodies is labeled as following; anti-CD3/PB, anti-CD4/1F:1P, anti-CD8/1F:5 P and anti-CD19/10F:1P.
  • The first step is to single stain with each marker, in this way the intensity of each label in the two detectors is know. Knowing the intensity from the single stain, and the amount and ratio of intensity between the two fluorochromes, the amount of each marker for any combination of markers can be solved using linear algebra. This sort of analysis, is simplified if no spillover is detected between the two fluorochromes, as well as not using the same labels on the markers that do bind to the same cell, however the latter is not a requirement for that analysis.
  • In this example the intensity measured can be addressed by the following; From the single stains we know the fluorescence from each marker as well as the relative intensity between the two detectors:
  • anti-CD3 is 0 in FITC, and P3 in PB ; where PB, and FITC are the respective detectors, and P and F are the fluorescence detected for the marker, and the ratio between fluorochromes are R3, thus;
  • anti-CD4 is F4 in FITC, and P4 in PB, and the ratio is R4
  • anti-CD8 is F8 in FITC, and P8 in PB, and the ratio is R8
  • anti-CD19 is F19 in FITC and P19 in, and the ratio is R19
  • Although, any combination of these could be solved in theory, only some are biological relevant, and the following equations for the intensity of each possible events is described as;
  •
    anti-CD3: FITC = 0 PB = P3
    anti-CD19: FITC = F19 PB = P19
    anti-CD3 + anti-CD4: FITC = F4 PB = P3 + P4
    anti-CD3 + anti-CD8: FITC = F4 + F8 PB = P3 + P8
  • Even if the amount of bound antibodies is different from that observed in the single stain, the system is solvable as the Ratio is know.
  •
    aCD3: FITC = a0 PB = aP3
    dCD19: FITC = dF19 PB = dP19
    aCD3 + bCD4: FITC = bF4 PB = aP3 + bP4
    aCD3 + eCD8: FITC = aF3 + eF8 PB = aP3 + e P8
  • It is straight forward to see how these can be solved knowing Fn and Pn and Rn for each marker, the only unknowns are “a, b, d and e”, thus 4 unknown and 4 equations are solvable. Software is used in real time to calculate these, and display the result as normal flow data, just in the same way as is done for compensation of spillover between fluorochromes.
  • The results can be visualized as any other dot plot in flow Cytometry. In this way reducing the amount of detectors and fluorochromes to be used in multi parameter flowcytometry.
    • Example 11
  • This is an example of how MHC multimers may be used for the detection of antigen specific T-cells simultaneously with activation of T cells.
  • In this example the following sub-processes are used:
  • Sampling: Blood retrieval from person using a vacutainer containing an anticoagulant
  • Sample preparation: Mononucleated cell fractioning using ficoll density centrifugation. Addition of labeling reagents. Cells in sample are stimulated.
  • Assaying: The samples are analysed using flow cytometry measuring fluorochrome labeled cells.
  • Data acquisition: Data is acquired using flow cytometry software and the described gating strategy.
  • Interpretation of data: The presence of antigen specific T cells secreting cytokine upon stimulation with cytokine in the sample is determined.
  • This example is a combination of i) direct detection of TCR, using MHC complexes coupled to any multimerisation as described elsewhere herein to stain antigen specific T cells, and ii) detection of induced intracellular cytokine production by addition of fluorophor-labelled anti-cytokine antibodies by flow cytometry.
  • Multicolor immunofluorescent staining with antibodies against intracellular cytokines and cell surface markers provides a high resolution method to identify the nature and frequency of cells which express a particular cytokine(s). In addition to enabling highly specific and sensitive measurements of several parameters for individual cells simultaneously, this method has the capacity for rapid analysis of large numbers of cells which are required for making statistically significant measurements.
  • Production of cytokines plays an important role in the immune response. Examples include the induction of many antiviral proteins by IFN-γ, the induction of T cell proliferation by IL-2 and the inhibition of viral gene expression and replication by TNF-α. Cytokines are not preformed factors; instead they are rapidly produced upon relevant stimulation. Intracellular cytokine staining relies upon the stimulation of T cells in the presence of an inhibitor of protein transport thus retaining the cytokines inside the cell.
  • Cellular activation to trigger cytokine production generally results in down-regulation of the T cell receptor. For this reason, MHC multimer staining is carried out prior to activation to ensure a good level of staining. The MHC multimers may be internalized with the T cell receptor during this period, but can still be detected in permeabilized cells. To analyze the effector function of antigen-specific T cells, the cells are first stained with MHC multimers, and then stimulated with antigen. This is followed by staining with antibodies specific for extracellular epitopes (such as CD8), then by membrane permeabilization and intracellular cytokine staining. The following protocol is an example of MHC multimer co-staining with anti-IFN-γ, TNF-α, MIP-1b, or IL-2.
  • Protocol applicable for intracellular staining of IFNg, TNFa, MIP-1b, or IL-2
    • 1. Blood is withdrawed from a donor using a vacutainer tube containing anticoagulant.
    • 2. Mononucleated cells are isolated using ficoll density centrifugation.
    • 3. Prepared peripheral blood cells are resuspended in phosphate buffered saline (PBS) at a cell concentration of 2×107 cells/ml.
  • 4. Transfer the cell suspension to individual tubes in 50 μl aliquots.
    • 3. Add relevant titrated fluorescently-labeled MHC multimers to the desired tubes, and incubate for 10 min at 22° C. (nonstimulated single-color controls should not be stained at this stage). Add 10 μl PBS to remaining tubes.
    • 4. Add 500 μl PBS to each tube. Centrifuge at 450×g for 5 minutes at 10° C.
    • 5. Aspirate supernatant. Agitate to disrupt cell pellets and resuspend in 200 μl complete RPMI.
    • 6. Dilute peptide/antigen stock 1:50 in complete RPMI. Add 2 μl of this (10 μg/ml (investigate the effect on cytokine response of titrating your peptide)) to each desired tube. If using Leukocyte Activation cocktail (LAC) as a control, rapidly thaw this at 37° C. in a water bath and add 0.33 μl of this to each desired tube.
  • 7. Place the tubes at 37° C. in a humidified CO2 incubator for 15 minutes to 1 hour.
    • 8. Add Brefeldin A (10 μg/ml final) to the desired tubes (n.b. LAC contains Brefeldin A) and return to the incubator. Incubate for 15 hours (the optimal incubation time is variable and must be determined).
    • 9. Remove tubes from the incubator. Centrifuge at 450×g for 5 minutes at 10° C.
    • 10. Aspirate supernatant. Resuspend desired cell pellets in 50 μl PBS containing an optimally titrated amount of anti-CD8 antibody. Add 50 μl PBS to remaining tubes. Note: Single-color controls should be stained at this stage. If additional phenotyping of samples is desired, antibodies to other cell surface receptors may also be added at this time.
    • 11. Incubate for 20 minutes on ice.
    • 12. Add 500 μl PBS to each tube. Centrifuge at 450×g for 5 minutes at 10° C.
    • 13. Aspirate supernatant. Agitate to disrupt cell pellets.
    • 14. Add 200 μl 4% paraformaldehyde to each sample tube. Vortex tubes. Incubate for 20 minutes on ice. This step will fix the cell morphology of the activated cells. Note: The procedure can be stopped at this point. Repeat steps 12 and 13. Resuspend the cells in 100 μl/tube PBS. Cover and store the cells at 4° C. for up to 3 days. To proceed, repeat steps 12 and 13. Resuspend the cells in 100 μl/tube permeabilization buffer and proceed to step 16.
    • 15. Add 200 μl permeabilization buffer to each tube.
    • 16. Centrifuge at 450×g for 5 minutes at 10° C. Aspirate supernatant.
    • 17. Add 100 μl permeabilization buffer to the sample tubes that are to be stained with anti-cytokine antibody. Add 100 μl PBS to the remaining tubes (i.e. Single-color controls).
    • 18. Incubate for 5 minutes at room temperature.
    • 19. Add an optimally titrated amount of conjugated anti-cytokine antibody to the desired sample tubes and mix.
    • 20. Incubate for 20 minutes at room temperature.
    • 21. Add 200 μl permeabilization buffer to each tube and centrifuge at 450×g for 5 minutes at 10° C. Aspirate supernatant and agitate tubes to disrupt the cell pellets.
    • 22. Resuspend the cells in 200 μl fix solution. Vortex tubes. It is important to vortex well when adding this fixative so that cells do not clump.
  • The samples are assayed on a flow cytometer and data acquired using appropriate software.
  • Sample is analysed using the following gating strategy: Gate on lymphocyte population if FCS/SSC plot. Select CD8 positive cells. If other gating reagents are included cells positive for these markers can be selected or deselected. Antigen specific T cells secreting cytokine is determined as MHC multimer, anti-cytokine double positive cells in the remaining population.
    • Example 12
  • This is an example of how MHC multimers may be used for the detection of antigen specific T-cells simultaneously with activation of T cells.
  • In this example the following sub-processes are used:
  • Sampling: Blood retrieval from person using a vacutainer containing an anticoagulant
  • Sample preparation: Mononucleated cell fractioning using ficoll density centrifugation. Addition of labeling reagents. Cells in sample are stimulated.
  • Assaying: The samples are analysed using flow cytometry measuring fluorochrome labeled cells.
  • Data acquisition: Data is acquired using flow cytometry software and the described gating strategy.
  • Interpretation of data: The presence of antigen specific T cells secreting cytokine upon stimulation with cytokine in the sample is determined.
  • This example is a combination of i) direct detection of TCR, using MHC complexes coupled as pentamers to stain antigen specific T cells, and ii) detection of induced intracellular cytokine production by addition of fluorophor-labelled anti-cytokine antibodies by flow cytometry. The antigenic origin is Epstein-Barr Virus (EBV), thus, immune monitoring of EBV infection
  • PBMCs were incubated with either a negative control (non-specific) Pentamer (A*0201/EBV (GLCTLVAML)) or a Pentamer specific for the cells of interest (B*0801/EBV (RAKFKQLL)), then stimulated with LAC (non-specific activation) or B*0801/EBV peptide (specific peptide activation) for 15 hours in the presence of Brefeldin A. Fixation, permeabilization and staining for IFN-γ were carried out exactly as detailed in the protocol outlined in example 11 above.
  • The samples are assayed on a flow cytometer and data acquired using appropriate software.
  • Sample was analysed using the following gating strategy: Gating on lymphocyte population in FCS/SSC plot. CD8 positive cells were selected. The presence of EBV specific T cells secreting IFN-γ were determined.
  • FIG. 9 illustrates Pentamer (specific or non-specific) versus intracellular IFN-γ staining after activation with specific or non-specific antigen.
  • This example shows that the MHC multimer constructs can be used to detect the presence of EBV specific T cells in the blood simultaneously with activation and intracellular staining of cytokines.
    • Example 13
  • In this example the following sub-processes are used:
  • Sampling: Blood retrieval from person using a vacutainer containing an anticoagulant and containing cell separating medium.
  • Sample preparation: Mononucleated cell fractioning using ficoll density centrifugation. Addition of labeling reagents.
  • Assaying: The samples are analysed using flow cytometry measuring fluorochrome labeled cells.
  • Data acquisition: Data is acquired using flow cytometry software and the described gating strategy.
  • 25
  • Interpretation of data: The presence of MHC dextramer specific T cells in the sample is determined and evaluated as a surrogate marker for Borrelia infection.
  • This is an example of how MHC multimers may be used for diagnosis of Lyme Disease in blood samples from humans infected with Borrelia bacteria.
  • In this example the MHC multimer used are MHC complexes coupled to fluorophor-labelled dextran (Dextramers). The dextramers are used for direct detection of TCR in flow Cytometry. The antigen origin is Borrelia, thus, immune monitoring of a Borrelia infection.
  • Lyme disease is caused by infection by Borrelia bacteria. During acute infection Borrelia specific activated T cells will be present in increased amounts in an activated state compared to healthy individuals. The presences of an increased amount of activated Borrelia specific T cells may thereby act as a surrogate marker for infection with Borrelia bacterium. MHC multimers carrying borrelia specific peptides is in this example used to detect the presence of Borrelia specific T cells in the blood of patients infected with Borrelia.
  • Purified MHC-peptide complexes consisting of HLA-A*0201 heavy chain, human beta2microglobulin and peptide derived from regions in Outer surface protein A or Flagellin B conserved among the three species Borrelia Burgdorferi, Borrelia Garinii and Borrelia Afzelii or a negative control peptide are generated by in vitro refolding, purified and biotinylated as described elsewhere herein. Biotinylated MHC-peptide complexes are then coupled to a 270 kDa dextran multimerization domain labelled with APC by interaction with streptavidin (SA) on the dextran multimerization domain. The dextran-APC-SA multimerization domain is generated as described elsewhere herein. MHC-peptide complexes are added in an amount corresponding to a ratio of three MHC-peptide molecules per SA molecule and each molecule dextran contains 3.7 SA molecule and 8.95 molecules APC. The final concentration of dextran is 3.8×10e-8 M.
  • The following MHC(peptide)/APC dextran constructs are made:
      • 1. APC-SA conjugated 270 kDa dextran coupled with HLA-A*0201 in complex with beta2microglobulin and the peptide ALIACKQNV derived from OspA.
      • 2. APC-SA conjugated 270 kDa dextran coupled with HLA-A*0201 in complex with beta2microglobulin and the peptide FTKEDTIT derived from OspA.
      • 3. APC-SA conjugated 270 kDa dextran coupled with HLA-A*0201 in complex with beta2microglobulin and the peptide SIQIEIEQL derived from Fla B
      • 4. APC-SA conjugated 270 kDa dextran coupled with HLA-A*0201 in complex with beta2microglobulin and the peptide NLNEVEKVL derived from Fla B
      • 5. APC-SA conjugated 270 kDa dextran coupled with HLA-A*0201 in complex with beta2microglobulin and the peptide SLAKIENAI derived from Fla B
      • 6. APC-SA conjugated 270 kDa dextran coupled with HLA-A*0201 in complex with beta2microglobulin and the non-sense peptide GLAGDVSAV
  • The binding of the above described MHC(peptide)/APC dextran is used to determine the presence of Osp A or Fla B specific T cells in the blood from Borrelia infected individuals by flow cytometry following a standard flow cytometry protocol.
  • Blood from a patient with Lyme disease is isolated and PBMC's purified by ficoll centrifugation separation. 100 ul of this purified blood is incubated with 10 μl of each of the MHC(peptide)/APC dextran constructs described above for 10 minutes in the dark at room temperature. 5 μl of each of each of the antibodies mouse-anti-human CD3/PB (clone UCHT1 from Dako), mouse-anti-human CD4/FITC (clone MT310 from Dako) and mouse-anti-human CD8/PE (clone DK25 from Dako) are added and the incubation continues for another 20 minutes at 4° C. in the dark. The samples are then washed by adding 2 ml PBS; pH=7.2 followed by centrifugation for 5 minutes at 300×g and the supernatant removed. The washing step is repeated twice. The washed cells are resuspended in 400-500 μl PBS+1% BSA; pH=7.2 and analyzed on flowcytometer. Cells were gated as follows: lymphocyte gate in FCS/SSC plot, removal of all CD4 positive cells. Cells labeled with anti-CD3/PB are positively selected.
  • In the remaining population of cells the presence of cells labeled with anti-CD8/PE and the MHC(peptide)/APC dextran constructs 1, 2, 3, 4 and 5 described above are Borrelia specific T cells. The presence of Borrelia specific T cells indicate that the patient are infected with Borrelia bacteria. Blood analysed with the negative control MHC(peptide)/APC dextran construct 6 show no staining of CD3 and CD8 positive cells with this MHC(peptide)/APC dextran construct. Results are shown in FIG. 10.
  • The presences of Borrelia antigen specific cells is a surrogate marker for the presence of ongoing Borrelia infection. Therefore this test is an indication of that the donor from which the sample derive have a Borrelia infection.
  • The sensitivity of the above described diagnostic test may be enhanced by addition of labeled antibodies specific for activation markers expressed in or on the surface of the Borrelia specific T cells.
    • Example 14
  • This is an example of how multiple marker molecules may be labeled with same label.
  • It is also a positive control experiment determining whether a sample contains or derive from a donor with certain MHC allele types.
  • In the present example the sample is analysed for the presences of T cells recognizing common pathogen antigens when bound to the MHC alleles HLA-A*0101, HLA-A*0201 and HLA-B*0702, thereby indirectly typing the sample for these three MHC alleles.
  • 106 HBPMC are stained with a mix of 9 MHC-Dextramers (3-10 μl of each), carrying antigenic peptides derived from the antigenic proteins Influenza MP1, EBV BMLF-1 and CMV pp65. 3 different HLA alleles are used HLA-A*0101, HLA-A*0201 and HLA-B*0702 each carrying peptides derived from the 3 antigenic proteins. All MHC-Dextramers are all labeled with APC.
  • The sample is incubated for 10 min, where after the sample is stained with 5 μl of each of the antibodies; CD14/FITC, CD19/FITC, CD3/RPE and CD8/RPE for 20 min.
  • Subsequently, the sample is washed and applied to flow cytomety analysis.
  • The sample is analyzed by sequential gating; the lymphocytes are identified using the scatter parameters, followed by applying an exclusion gate defined by the CD14/FITC, CD19/FITC positive cells. In the remaining cell populations the cytotoxic T cell population is defined by being RPE positive (CD3 and CD8 positive).
  • It is now analyzed if there is one or more population(s) of APC positive cells. If a population of cells is identified we can conclude that the donor has one or more of the tissue types HLA-A*0101, HLA-A*0201 and HLA-B*0702. If no population is formed we can conclude that either the tissue type is diverging from those in the test reagent, or the donor has no antigen specific cytotoxic T cells against the Flue-MP1, EBV-BMLF-1, CMV-pp65 antigens.
    • Example 15
  • This is an example of how multiple marker molecules may be labeled with same label.
  • This an example of a positive control experiment determining whether a sample contains or derive from a donor with certain MHC allele types.
  • In the present example the sample is analysed for the presences of T cells recognizing epitopes from an antigen from a common pathogen when bound to the MHC alleles HLA-A*0201, HLA-A*2402 and HLA-B*0702, thereby indirectly typing the sample for these three MHC alleles.
  • The following MHC dextramer constructs were made:
      • 1. APC-SA conjugated 270 kDa dextran coupled with HLA-A*0201 in complex with beta2microglobulin and the peptide NLVPMVATV derived from CMV pp65.
      • 2. APC-SA conjugated 270 kDa dextran coupled with HLA-A*2402 in complex with beta2microglobulin and the peptide QYDPVAALF derived from CMV pp65.
      • 3. APC-SA conjugated 270 kDa dextran coupled with HLA-B*0702 in complex with beta2microglobulin and the peptide TPRVTGGGAM derived from CMV pp65
  • 106 HBPMC are stained with a mix of the 3 MHC-Dextramers constructs 1, 2, 3 described above (3-10 μl of each).
  • The sample is incubated for 10 min, where after the sample is stained with 5 μl of each of the antibodies; CD14/FITC, CD19/FITC, CD3/RPE and CD8/RPE for 20 min.
  • Subsequently, the sample is washed and applied to flow cytomety analysis.
  • The sample is analyzed by sequential gating; the lymphocytes are identified using the scatter parameters, followed by applying an exclusion gate defined by the CD14/FITC, CD19/FITC positive cells. In the remaining cell populations the cytotoxic T cell population is defined by being RPE positive (CD3 and CD8 positive).
  • It is now analyzed if there is one or more population(s) of APC positive cells. If a population of cells is identified we can conclude that the donor has one or more of the tissue types HLA-A*0201, HLA-A*2402 or HLA-B*0702. If no population is formed we can conclude that either the tissue type is diverging from those in the test reagent, or the donor has no antigen specific cytotoxic T cells against the, CMV-pp65 antigen.
    • Example 16
  • This is an example of how to type a sample for the presence of certain MHC alleles using MHC allele specific antibodies in a standard flow cytometry experiment.
  • HPBMC from the blood of two donors, donor 1 and 2 were isolated by a standard protocol using Ficoll-Hypaque. A fraction of the cells from each sample was transferred to two new tube making a total of 4 tubes. 10 μl PE-labeled antibody specific for the MHC alleles HLA-A*02 (clone BB7.2 from BD) was added to one tube for each sample and biotinylated antibody specific for HLA-A*03 (ab31572 from abcam) was added to the second tube of each sample. Samples were incubated at 4° C. for 10 minutes in the dark. Then 10 μl SA/PE (R0438 from Dako) was added to tubes with anti-HLA-A*03 antibody and incubation continued for another 30 minutes.
  • Cells were washed in 2 ml PBS, centrifuged and supernatant removed. Cells were resuspended I PBS and samples analysed on a CyAn flow cytometer. As a control unstained cell samples from each donor was also analysed.
  • Cells were gated using a lymphocyte gate in a FCS/SSC plot and the presence of PE positive staining was determined in each sample.
  • Donor 1 was positive for HLA-A*02 and negative for HLA-A*03 and donor 2 was negative for HLA-A*02 and positive for HLA-A*03. Results are shown in FIG. 11.
  • The results was confirmed in a PCR test using probes specific for HLA-A*02 and HLA-A*03 (data not shown).
  • This example demonstrates that antibodies specific for a specific HLA allele can be used to determine the HLA type of cells in a given sample.
    • Example 17
  • This example describes how to identify specific T cells in a blood sample with MHC multimers using flow cytometry analysis without lysis of red blood cells and without washing the cells after staining. MHC complexes in this example consisted of HLA-A*0201 heavy chain, human beta2microglobulin and different peptides, and the MHC complexes were coupled to a 270 kDa dextran multimerization domain.
  • Purified MHC-peptide complexes consisting of human heavy chain, human beta2microglobulin and peptide were generated by in vitro refolding, purified and biotinylated as described elsewhere herein. Biotinylated MHC-peptide complexes were then coupled to a 270 kDa dextran multimerization domain labelled with PE by interaction with streptavidin (SA) on the dextran multimerization domain. The SA-PE-dextran was made as described elsewhere herein. MHC-peptide complexes was added in an amount corresponding to a ratio of three MHC-peptide moleculess per SA molecule and each molecule dextran contained 6.1 SA molecule and 3.9 molecules PE. The final concentration of dextran was 3.8x10e-8 M. The following constructs were made:
  • 1. PE conjugated 270 kDa dextran coupled with HLA-A*0101 in complex with beta2microglobulin and the peptide VTEHDTLLY derived from Human Cytomegalo Virus (HCMV).
      • 2. PE conjugated 270 kDa dextran coupled with HLA-A*0101 in complex with beta2microglobulin and the peptide IVDCLTEMY derived from ubiquitin specific peptidase 9 (USP9).
      • 3. PE conjugated 270 kDa dextran coupled with HLA-A*0201 in complex with beta2microglobulin and the peptide NLVPMVATV derived from Human Cytomegalo Virus (HCMV).
      • 4. PE conjugated 270 kDa dextran coupled with HLA-A*0201 in complex with beta2microglobulin and the peptide ILKEPVHGV derived from Human Immunodeficiency Virus (HIV).
      • 5. PE/SA conjugated 270 kDa dextran coupled with HLA-B*0207 in complex with beta2microglobulin and the peptide TPRVTGGGAM derived from Human Cytomegalo Virus (HCMV).
      • 6. PE conjugated 270 kDa dextran coupled with HLA-B*0207 in complex with beta2microglobulin and the peptide RPHERNGFTVL derived from Human Cytomegalo Virus (HCMV).
      • 7. PE conjugated 270 kDa dextran coupled with HLA-B*0207 in complex with beta2microglobulin and the peptide TPGPGVRYPL derived from Human Immunodeficiency Virus (HIV).
  • These seven MHC multimer constructs were used for detection of specific T cells in flow cytometry analysis using a no-lyse no-wash procedure. Blood samples from three individual donors were analyzed. The donors had previously been screened for the presence of specific T cells using a general staining procedure including lysis and wash of the cell sample, and donor one turned out to be positive for HLA*0201 in complex with the peptide NLVPMVATV, donor two were positive for HLA*0101 in complex with the peptide VTEHDTLLY and donor three were positive for HLA-B*0207 in complex with the peptides TPRVTGGGAM and RPHERNGFTVL. In this experiment blood from each donor were analyzed with the MHC multimer construct they were supposed to have specific T cells restricted for and with MHC multimers of same haplotype but carrying a negative control peptide. The negative control peptides were either derived from HIV or the self-protein USP 9. Self-protein here means a naturally occurring protein in normal cells of a human individual. Normal healthy donors not infected with HIV are not expected to have specific T cells recognizing HIV derived peptides or peptides derived from self-proteins in complex with any HLA molecule in an amount detectable with this analysis method.
  • The blood were stained as follows:
  • 100 μl EDTA stabilized blood were incubated with 5 μl MHC(peptide)/PE dextran for 5 minutes at room temperature. Anti-CD45/PB, anti-CD3/FITC and anti-CD8/APC antibody in an amount of 0.4-1.2 μg/sample was added to each tube and the incubation continued for another 15 minutes. 850 μl PBS; pH=7.2 was added and the sample analyzed on a CyAn ADP flowcytometry instrument with a speed of 150 μl/minute. A total of 20.000 CD8 positive cells were acquired. During analysis CD45/PB antibody was used to set a trigger discriminator to allow the flow cytometer to distinguish between red blood cells and stained white blood cells (see FIG. 12A). Furthermore CD3/FITC antibody was used to select CD3 positive cells in a second gating strategy (see FIG. 12B).
  • Blood from donor one showed specific staining with HLA-A*0201(NLVPMVATV) multimer (construct 3) while no staining of specific T cells was observed with the negative control HLA-A*0201(ILKEPVHGV) multimer (construct 4). Donor two showed specific staining with HLA-A*0101(VTEHDTLLY) multimer (construct 1) and no staining was observed with the negative control HLA-A*0101(IVDCLTEMY) multimer (construct 2). In blood from donor three a population of T cells were stained with HLA-B*0207(TPRVTGGGAM) multimer (construct 5) and another population with HLA-B*0207(RPHERNGFTVL) multimer (construct 6) while no specific staining was observed with the negative control HLA-B*0207(TPGPGVRYPL) multimer (construct 7). The results are shown in FIG. 13.
  • We have shown that MHC multimers of three different haplotypes can be used to identify specific T cells in blood samples from three different donors using an approach without lysing red blood cells and without wash following staining with MHC multimer.
  • This method is simple, fast and interfere as little as possible with cells in the blood sample.
    • Example 18
  • This example illustrates how MHC multimers together with counting beads was used for exact numeration of MHC-peptide specific T cells in a flow cytometry analysis whit no lyses of red blood cells and no washing steps during or after staining. Counting beads in this example was CytoCount™, Count Control Beads from Dako that are polystyrene Fluorospheres with a diameter of 5.2 μm. The MHC multimer consisted of HLA-A*0101 heavy chain complexed with human beta2microgloblin and a peptide and the MHC-peptide complexes were coupled to a 270 kDa dextran multimerization domain labelled with PE. MHC multimers were generated as described elsewhere herein and the following two constructs were made:
      • 1) PE conjugated 270 kDa dextran coupled with HLA-A*0101 in complex with beta2microglobulin and the peptide VTEHDTLLY derived from Human Cytomegalo Virus (HCMV).
      • 2) PE conjugated 270 kDa dextran coupled with HLA-A*0101 in complex with beta2microglobulin and the peptide IVDCLTEMY derived from ubiquitin specific peptidase 9 (USP9).
  • Construct 2 is a negative control for construct 1 in this example and both were used for detection of specific T cells by flow cytometry using a no-lyse no-wash procedure: 100 μl of EDTA stabilized blood from a donor positive for HLA*0101 in complex with the peptide VTEHDLLY were incubated with 5 μl MHC multimer for 5 minutes at room temperature. Anti-CD45/CY, anti-CD3/PB and anti-CD8/APC antibody in an amount of 0.4-1.2 μg/sample was added and the incubation continued for another 15 minutes. 850 μl PBS; pH=7.2 was added together with precise 50 μl CytoCount beads 1028 bead/μl and the sample analyzed on a CyAn ADP flowcytometry instrument with a speed of 150 μl/minute. A total of 20.000 CD8 positive cells were acquired. During analysis CD45/CY antibody was used to set a trigger discriminator to allow the flow cytometer to distinguish between red blood cells and stained white blood cells.
  • A dot plot was made for each sample showing MHC multimer vs CD8 positive events (se FIGS. 14A and B). Based on the negative control a gate comprising events representing CD8 positive T cells specific for MHC multimer was defined. Similarly histogram plots for each sample was made showing FITC signal vs counts (FIGS. 14C and D). In these histograms the amount of beads in the analyzed sample were identified since the beads in contrast to the cells emit light in the FITC channel. In principle the beads could be visualized in any fluorochrome channel because they emit light in all channels but it was important to visualize the beads in a channel where there was no interfering signal from labelled cells.
  • The concentration of T cells specific for HLA-A*0101(VTEHDTLLY) multimer (construct 1) in the blood sample were determined using the counting beads as an internal standard. Events obtained from staining with the negative control MHC multimer, construct 2, were defined as background signals and subtracted from the result obtained from staining with construct 1.

  • Concentration of HLA-A*0101(VTEHDTLLY) specific T cells in the blood sample=((Count of MHC multimer+CD8+ positive cells, construct 1×concentration of beads×dilution factor of beads)/counted beads))−((Counted MHC multimer+CD8+ cells, construct 2×concentration of beads x dilution factor of beads)/counted beads)=992.6 cells/ml
  • For details se FIG. 14.
  • This experiment demonstrated how CytoCount™ counting beads together with MHC multimers could be used to determine the exact concentration of MHC-peptide specific T cells in a blood sample using a no-lyse no-wash method.
    • Example 19
  • Procedure of Immune Diagnostic Assay Performed on Whole Blood by ELISA Based Method.
  • This is an example of indirect detection of the presence of T cells, where cells in suspension are stimulated/induced to procure cytokines. The cytokine production is detected by a chromogen assay using anti-cytokine antibodies. The antigenic peptide origin is Tuberculosis (TB), thus, immune monitoring of TB infection.
  • The ELISA method on whole blood is performed by the use of the QuantiFERON-CMI (QF-CMI, manufactured by Cellestis Limited. South Melbourne, Australia.) Step 2 is performed according to the protocol and with reagents provided by the manufacture.
  • 0.5 ml of blood is used instead of 1 ml, and therefore a 48-well plate instead of a 24-well is used. The whole procedure of the test requires the use of the following materials and reagents: IFN-gamma QuantiFERON-CMI kit; 48-well plate and 7 tubes, each containing the different stimuli at the desired concentration. The ELISA technique, performed on whole blood, is composed of the following steps:
  • Step 1. Culture of Whole Blood
      • 1. mix the tubes containing heparinised whole blood
      • 2. distribute blood (500 μl/well) into sterile 48-well plaies. For the children similar results can be obtained using 250 μl/well of blood. Consequently the volume of the Reagents 1-7 to be added will be 50% of the volume indicated.
      • 3. add control mitogen and specific antigens according to the table below.
      • 4. mix well.
      • 5. incubate plate at 37° C. for 24 hours
      • 6. harvest plasma aliquots from each well
  • TABLE
    Stimulus and working Concentration of Volume
    concentration (values in stock solution to be
    parentheses relate to for 0.5 ml of added
    Reagent concentration in μg/mL) blood (mg/mL) (μL)
    1 CTR* or DMSO (8)**, ** 50
    or DMSO (60)**
    2 ESAT-6 protein (0.2) and 0.01 50
    CFP-10 protein (0.2)
    3 ESAT-6 pooled peptides (50) 0.5 50
    4 CFP-10 pooled peptides (8) 0.08 50
    5 ESAT-6 and CFP-10 pooled 0.6 50
    peptides (58)
    6 PHA (1) 0.05 50
    7 PPD (5) 0.05 50
    *CTR: complete culture medium
    **for DMSO: an identical solvent concentration will be added in reagent 1 corresponding to the amount of DMSO present in reagents 3-5.
  • Step 2. IFN-Gamma-Based ELISA
      • 1. prepare “conjugated antibody” by dissolving it in the “green diluent” solution, and distribute in the ready-to-use ELISA plate.
      • 2. add harvested plasma and “standard solution” to the corresponding wells containing “green diluent”.
      • 3. mix well.
      • 4. cover the plate and incubate for 2 hours at room temperature.
      • 5. wash with “wash buffer”.
      • 6. prepare the 100× “chromogen” by diluting it with the “enzyme substrate buffer” and distribute in the plate.
      • 7. cover the plate and incubate for 30 minutes at room temperature in the dark.
      • 8. add stop solution to block the reaction and immediately read optic density in each well, at 450/620 nm using an ELISA reader.
  • Evaluation of Test Results and Diagnostic Response:The optic density values of the plate are analysed, a standard curve and IFN-gamma values, expressed as International Units (I.U.)/mL, are calculated for each well by the use of special software provided by the manufacturer.
    • Example 20
  • This is an example of measurement of antigen reactive T-Cells by IFN-γ capture in blood samples by ELISPOT assay.
  • This is an example of indirect detection of T cells, where individual cells are immobilized and measured by a chromogen assay.
  • The example provides a sensitive assay for the detection of T-cells reactive to an antigen by detecting a soluble factor whose secretion is induced by stimulation of the T-cell by the antigen.
  • A summary flow chart of the method is shown in FIG. 15. In brief, peripheral blood is diluted threefold in Dulbecco's phosphate buffered saline (DPBS), underlain with 15 ml of Ficoll (Pharmacia Ficoll-Paque #17-0840-02, Piscataway, N.J.) per 40 ml diluted blood in a 50 ml polypropylene centrifuge tube, and spun at 2000 RPM for 20 minutes in a Beckman CS-6R centrifuge (Beckman Inc., Palo Alto, Calif.). The buffy layer at the DPBS/Ficoll interface is removed, washed twice with DPBS and once with human tissue culture medium (hTCM: αMEM+5% heat inactivated human AB serum (Ultraserum, BioWhittaker, Walkersville, Md.), penicillin/streptomycin, 1-glutamine) at low RCF to remove platelets. Sixty percent of the PBMCs are resuspended in freezing medium (10% dimethyl sulfoxide(Sigma Chenical Co., St. Louis, Mo.), 90% fetal bovine serum to a concentration of 5×106 cells/ml, frozen in a programmable Cryo-Med (New Baltimore, Mich.) cell freezer, and stored under liquid nitrogen until needed.
  • The purified PBMCs are plated at 2×105 cells/well at a volume of 0.1 ml in 96 well Costar cell culture plates. An equal volume of antigen at 10 μg/ml is added to triplicate or sextuplet sets of wells and the plate is incubated in a 37° C., 5% CO2 incubator. On day five, 10 μl/well of 100 U/ml stock recombinant IL-2 (Advanced Biotechnologies Inc., Columbia, Md.) is added to each well. On day 8, frozen PBMCs are thawed, washed in DPBS+0.5% bovine serum albumin (BSA) to remove DMSO, resuspended to a concentration of 4×106 cells/ml in hTCM, and γ-irradiated (3,000 RADS). Fifty microliters/well are dispensed along with 50 μl of the appropriate antigen at a stock concentration of 40 μl/ml to give a final antigen concentration of 10 μg/ml.
  • To prepare a capture plate, IFN-γ capture antibody (monoclonal mouse anti-human IFN-g, Endogen #M700A, Cambridge, Mass.) is diluted to 10 μg/ml in sterile 0.1 M Na(CO3)2 pH 8.2 buffer, aliquotted at 50 μl/well in flat bottomed 96 well sterile microtiter plates (Corning Costar Corp.), and incubated at 4° C. for a minimum of 24 hours. Prior to use, excess antibody is removed and wells are washed twice with dPBS+1% Tween 20 (PBST). To block further nonspecific protein binding, plates are incubated with 250 μl/well of PBS+5% BSA at room temperature for 1 hour. After discarding the blocking solution, wells are washed once with PBST (0.1% Tween), followed by hTCM in preparation for the antigen stimulated cells.
  • On day 9 of the assay, twenty four hours after the second antigen stimulation, the stimulation plate is spun for 5 minutes at 1500 RPM in a Beckman CS-6R centrifuge and 90 μl of supernatant is carefully removed from each well with a micropipette. The pelleted cells are resuspended in 100 μl of hTCM, pooled in sterile tubes (Corning Costar corp sterile ClusterTAb #4411, Cambridge, Mass.), mixed and transferred into an equal number of wells of an anti IFN-γ capture plate. Capture plates are incubated undisturbed at 37° C. for 16-20 hours. At the end of the IFN-γ secretion phase, the cells are discarded and the plates are washed three times with 0.1% PBST. A final aliquot of PBST is added to the wells for ten minutes, removed, and 100 μl of a 1:500 dilution of rabbit anti-human IFN-γ polyclonal antibody (Endogen #P700, Cambridge, Mass.) in PBST+1% BSA is added to each well for 3.5 hours at room temperature with gentle rocking. Unbound anti-IFN-y polyclonal antibody is removed by three washes with PBST, followed by a wash with 250 μl of 1× Tris-buffered saline+0.05% Tween 20 (TBST). Next, a 100 μl aliquot of 1:5000 alkaline phosphatase-conjugated mouse anti-rabbit polyclonal antibody (Jackson Immunological #211-055-109, West Grove, Pa.) diluted in TBST is added to each well and incubated at room temperature for 1.5-2 hours with gentle rocking. Excess enzyme-conjugated antibody is removed by three washes with PBST and two washes with alkaline phosphatase buffer (APB=0.1 M NaCl, 0.05 M MgCl.sub.2, 0.1 M Tris HCl, pH 9.5) followed by addition of the substrate mix of p-Toluidine salt and nitroblue tetrazolium chloride (BCIP/NBT, GIBCO BRL #18280-016, Gaithersburg, Md.). To stop the calorimetric reaction, plates were washed three times in dH2O, inverted to minimize deposition of dust in the wells, and dried overnight at 28° C. in a dust free drying oven.
  • Images of the spots corresponding to the lymphokine secreted by individual antigen-stimulated T cells are captured with a CCD video camera and the image is analyzed by NIH image software. Captured images are enhanced using the Look Up Table which contrasts the images. Thresholding is then applied to every image and a wand tool is used to highlight the border to effectively subtract the edge of the well so that background counts won't be high and artificial. Density slicing over a narrow range is then used to highlight the spots produced from secreting cells. Pixel limits are set to subtract out small debris and large particles, and the number of spots falling within the prescribed pixel range are counted by the software program. Totals from each well are then manually recorded for future analysis. Alternatively, spots can be counted by other commercially available or customized software applications, or may be quantitated manually by a technician using standard light microscopy. Spots can also be counted manually under a light microscope.
  • The protocol detailed above can be used for the enumeration of single IFN-y secreting T cells with any specificity.
    • Example 21
  • This is an example of measurement of antigen reactive T-Cells by IFN-γ capture in blood samples from Multiple Sclerosis (MS) patients by ELISPOT.
  • This is an example of indirect detection of T cells, where individual cells are immobilized and measured by a chromogen assay.
  • The antigenic peptide origin is MS, thus, immune monitoring of MS.
  • The example provides a sensitive assay for the detection of T-cells reactive to the antigen Myelin Basic Protein (MBP), by detecting a soluble factor whose secretion is induced by stimulation of the T-cell by the antigen.
  • This example is similar to the experiment above. PBMCs from Multiple Sclerosis patients are isolated, prepared and stored as described in the example above.
  • The purified PBMCs are plated at 2×105 cells/well at a volume of 0.1 ml in 96 well Costar cell culture plates. An equal volume of antigen, MBP 83-102 (YDENPWHFF KNIVTPRTPP) or 144-163 (VDAQGTLSKIFKLGGRDSRS), at 10 μg/ml is added to triplicate or sextuplet sets of wells and the plate is incubated in a 37° C., 5% CO2 incubator. On day five, 10 μl/well of 100 μ/ml stock recombinant IL-2 is added to each well. On day 8, frozen PBMCs are thawed, washed in DPBS+0.5% BSA to remove
  • DMSO, resuspended to a concentration of 4×106 cells/ml in hTCM, and γ-irradiated (3,000 RADS). 50 μl/well are dispensed along with 50 μl of the appropriate antigen at a stock concentration of 40 μl/ml to give a final antigen concentration of 10 μg/ml.
  • A capture plate with IFN-γ antibody is prepared, washed and blocked as described in the example above.
  • On day 9 of the assay, twenty four hours after the second antigen stimulation, the stimulation plate is spun for 5 minutes at 1500 RPM and 90 μl of supernatant is carefully removed from each well with a micropipette. The pelleted cells are resuspended in 100 μl of hTCM, pooled in sterile tubes, mixed and transferred into an equal number of wells of an anti IFN-γ capture plate. Capture plates are incubated undisturbed at 37° C. for 16-20 hours. At the end of the IFN-γ secretion phase, the cells are discarded and the plates are washed three times with 0.1% PBST. A final aliquot of PBST is added to the wells for ten minutes, removed, and 100 μl of a 1:500 dilution of rabbit anti-human IFN-γ polyclonal antibody in PBST+1% BSA is added to each well for 3.5 hours at room temperature with gentle rocking. Unbound anti-IFN-γ polyclonal antibody is removed by three washes with PBST, followed by a wash with 250 μl of 1× Tris-buffered saline+0.05% Tween 20 (TBST). Next, a 100 μl aliquot of 1:5000 alkaline phosphatase-conjugated mouse anti-rabbit polyclonal antibody diluted in TBST is added to each well and incubated at room temperature for 1.5-2 hours with gentle rocking. Excess enzyme-conjugated antibody is removed by three washes with PBST and two washes with alkaline phosphatase followed by addition of the substrate mix of p-Toluidine salt and nitroblue tetrazolium chloride. To stop the calorimetric reaction, plates were washed three times in dH2O, inverted to minimize deposition of dust in the wells, and dried overnight at 28° C. in a dust free drying oven.
  • Images of the spots corresponding to the lymphokine secreted by individual antigen-stimulated T cells are captured with a CCD video camera and the image is analyzed as described in the example above
  • We conclude that the experiment detailed above can be used for the enumeration of single IFN-γ secreting T cells in blood from Multiple Sclerosis patients.
    • Example 22
  • This is an example of measurement of antigen reactive T-Cells by IFN-γ capture in blood samples from Melanoma patients by ELISPOT.
  • This is an example of indirect detection of T cells, where individual cells are immobilized and measured by a chromogen assay.
  • The antigenic peptide origin is Melanoma, thus, immune monitoring of cancer.
  • The example provides a sensitive assay for the detection of T-cells reactive to the antigen MELAN-A/MART-1 27-35, by detecting a soluble factor whose secretion is induced by stimulation of the T-cell by the antigen.
  • This example is similar to the experiment above. PBMCs from Melanoma patients are isolated, prepared and stored as described in the example above.
  • The purified PBMCs are plated at 2×105 cells/well at a volume of 0.1 ml in 96 well Costar cell culture plates. An equal volume of antigen, MELAN-A/MART-1 27-35 (AAGIGILTV), at 10 μg/ml is added to triplicate or sextuplet sets of wells and the plate is incubated in a 37° C., 5% CO2 incubator. On day five, 10 μl/well of 100 U/ml stock recombinant IL-2 is added to each well. On day 8, frozen PBMCs are thawed, washed in DPBS+0.5% BSA to remove DMSO, resuspended to a concentration of 4×106 cells/ml in hTCM, and y-irradiated (3,000 RADS). 50 μl/well are dispensed along with 50 μl of the appropriate antigen at a stock concentration of 40 μl/ml to give a final antigen concentration of 10 μg/ml.
  • A capture plate with IFN-γ antibody is prepared, washed and blocked as described in the example above.
  • On day 9 of the assay, twenty four hours after the second antigen stimulation, the stimulation plate is spun for 5 minutes at 1500 RPM and 90 μl of supernatant is carefully removed from each well with a micropipette. The pelleted cells are resuspended in 100 μl of hTCM, pooled in sterile tubes, mixed and transferred into an equal number of wells of an anti IFN-γ capture plate. Capture plates are incubated undisturbed at 37° C. for 16-20 hours. At the end of the IFN-γ secretion phase, the cells are discarded and the plates are washed three times with 0.1% PBST. A final aliquot of PBST is added to the wells for ten minutes, removed, and 100 μl of a 1:500 dilution of rabbit anti-human IFN-γ polyclonal antibody in PBST+1% BSA is added to each well for 3.5 hours at room temperature with gentle rocking. Unbound anti-IFN-γ polyclonal antibody is removed by three washes with PBST, followed by a wash with 250 μl of 1× Tris-buffered saline+0.05% Tween 20 (TBST). Next, a 100 μl aliquot of 1:5000 alkaline phosphatase-conjugated mouse anti-rabbit polyclonal antibody diluted in TBST is added to each well and incubated at room temperature for 1.5-2 hours with gentle rocking. Excess enzyme-conjugated antibody is removed by three washes with PBST and two washes with alkaline phosphatase followed by addition of the substrate mix of p-Toluidine salt and nitroblue tetrazolium chloride. To stop the calorimetric reaction, plates were washed three times in dH2O, inverted to minimize deposition of dust in the wells, and dried overnight at 28° C. in a dust free drying oven.
  • Images of the spots corresponding to the lymphokine secreted by individual antigen-stimulated T cells are captured with a CCD video camera and the image is analyzed as described in the example above
  • We conclude that the experiment detailed above can be used for the enumeration of single IFN-γ secreting T cells in blood from Melanoma patients.
    • Example 23
  • This is an example of how MHC tetramers may be used to monitor the immune status of a patient following transplantation, thereby guiding the immune suppressive treatment. The detection method used is a direct detection of individual specific T cells using flow cytometry.
  • T lymphocytes (T cells) play a critical role in host immune defense to microbial infections. Specialized phagocytic cells, known as antigen-presenting cells (APCs), process foreign proteins resulting in peptide fragment expression on their cell surfaces. These peptides are complexed to MHC (major histocompatibility complex) molecules. When naïve T cells encounter APCs expressing foreign peptide-MHC molecules, the T cells are induced to differentiate and proliferate. The result is a cellular immune response consisting of T cells that are capable of recognizing and destroying infected cells expressing the specific peptide—MHC molecule. Long-term memory cells capable of rapidly responding to a repeat infection are also generated.
  • These antigen-specific T cells can be detected using MHC Tetramers.
  • Within their lifetime, more than half of the world's population will become infected with human cytomegalovirus (CMV), a member of the ubiquitous herpes virus family.
  • Once an individual is infected, viral particles can escape total immunoclearance and remain dormant within the host's cells. CMV antigen-specific CD8+ cytotoxic T cells can control the latent CMV infection in healthy individuals.
  • The detection of CD8+ antigen-specific T cells requires cognate recognition of the T cell receptor (TCR) by a unique combination of a Class I major histocompatibility complex (MHC) molecule coupled with a specific antigen peptide. Antigen-specific TCR on the surface of CD8+ T cells is recognized by MHC Class I Tetramers MHC Class I Tetramers are a complex of four peptide—MHC Class I molecules stably bound with streptavidin to which a fluorochrome (most often PE) is attached. MHC Class I Tetramers are used in combination with fluorescently conjugated CD3 and CD8 antibodies to determine the frequency of CD3+ CD8+ T cells.
  • The following flow cytometric protocol describes a two-panel technique for the determination of the absolute count (cells/μL) of CMV antigen-specific CD8+ T cells in whole blood. Panel 1 consists of a single assay tube containing whole blood, anti-CD3, anti-CD4, anti-CD8 monoclonal antibody reagents, and Flow-Count Fluorospheres and is prepared using a lyse-no-wash method. Results from panel 1 provide a direct determination of the CD3+CD4+ and CD3+CD8+ T cell subset absolute counts. Panel 2 consists of a variable number of tubes dependent on the MHC or HLA (Human Leukocyte Antigen) phenotype of the individual being tested. Each tube in panel 2 contains whole blood, anti-CD3, anti-CD8, and specific CMV MHC Class I Tetramer and is prepared using a lyse-with-wash method. Results from panel 2 are used to determine the relative percentage of antigen-specific CD3+CD8+ T cells present within a sample.
  • Identification of the CD3+CD8+ population is common to both panels. Therefore, by applying the percentage of antigen-specific CD3+CD8+ T cells determined in panel 2 to the absolute CD3+CD8+ cell count determined in panel 1, the absolute number of antigen-specific CD3+CD8+ T cells is determined per μL of whole blood. Both panels may be prepared concomitantly. Control Cells are prepared as described above for use in panel 1 only.
  • Detection and Enumeration of CMV Antigen-Specific CD8-Positive T Lymphocytes in Whole Blood by Flow Cytometry.
  • Reagent Preparation
    • 1. Lyse/Fixative solution: Calculate the total volume of Lyse/Fixative solution required (panel 1—1 mL/tube. Add 25 μL of MHC Tetramer Fixative Reagent to 1 mL of iTAg MHC Tetramer Lyse Reagent.
    • 2. 0.1% formaldehyde in PBS: Calculate the total volume of 0.1% formaldehyde/PBS fixative solution required (panel 2 only—0.5 mL/tube). Add 12.5 μL iTAg MHC Tetramer Fixative Reagent to 1 mL of PBS.
    • 3. Bring all monoclonal antibody reagents, and MHC Tetramers to room temperature (RT) before pipetting. Vortex before use.
    • 4. On the same day of data acquisition by flow cytometry, remove Flow-Count™ Fluorospheres from 4° C. storage. Bring to RT prior to use. Vortex for 10-12 seconds and avoid excessive mixing to minimize air bubble formation.
  • Panel 1. Determination of Absolute T Cell Counts—Lyse-no-Wash
    • 1. Appropriately label tubes for each patient being tested.
    • 2. Pipette 10 μL of anti-CD3, 10 μL of anti-CD4, and 10 μL of anti-CD8 into the bottom of each tube.
    • 3. Pipette 100 μL of whole blood into the bottom of each tube.
    • 4. Vortex gently to ensure complete mixing of whole blood sample with antibody reagents.
    • 5. Incubate tubes at room temperature (18-25° C.) for 20-30 minutes, protected from light.
    • 6. Add 1 mL of Lyse/Fixative solution to each tube and vortex immediately for one second after each addition.
    • 7. Incubate at room temperature for at least 10 minutes, protected from light.
    • 8. Store prepared samples at 4° C., protected from light, until the addition of Flow-Count Fluorospheres within 24 hours.
    • 9. Pipette 100 μL of adequately mixed room-temperature Flow-Count Fluorospheres into each tube immediately prior to analysis by flow cytometry.
    • 10. Vortex each tube for 5 seconds to ensure proper mixing and resuspension of cells and fluorospheres.
    • 11. Samples must be analyzed within one hour. Repeat vortexing immediately prior to flow-cytometric acquisition.
  • Panel 2. Determination of Relative Percent of CMV Antigen-Specific CD8+ T Cells—Lyse-with-Wash
    • 1. Appropriately label tubes for each patient being tested.
    • 2. Add 10 μL of specific MHC Class I Tetramer or Negative Tetramer and 10 μL each of anti-CD3 and anti-CD8 monoclonal antibody reagents into each tube.
    • 3. Add 200 μL of whole blood into each tube. Specimens with low leukocyte counts (<3.0×103/μL) or low lymphocyte counts (<0.5 ×103/μL) may require whole blood volumes up to 400 μL. Under these circumstances, up to 4 mL of Lyse/Fixative solution is required—all other reagent volumes remain as described.
    • 4. Vortex gently.
    • 5. Incubate at room temperature (18-25° C.) for 20-30 minutes, protected from light.
    • 6. Add 2 mL of Lyse/Fixative solution to each tube and vortex immediately for one second after each addition.
    • 7. Incubate at room temperature for at least 10 minutes, protected from light.
    • 8. Centrifuge tubes at 150×g for 5 minutes.
    • 9. Aspirate or decant the supernatant.
    • 10. Add 3 mL of PBS.
  • 11. Centrifuge tubes at 150×g for 5 minutes.
    • 12. Aspirate or decant the supernatant.
    • 13. Resuspend the cell pellet in 500 μL of PBS with 0.1% formaldehyde.
    • 14. Vortex each tube for 5 seconds.
    • 15. Store prepared samples at 4° C. protected from light for a minimum of 1 hour (maximum 24 hours) until analysis by flow cytometry.
  • Samples are analysed on flowcytomer and data sets for both panels acquired.
  • MHC negative tetramer is used to assess the level of background PE fluorescence and non-specific binding for all MHC I tetramers conjugated to SA-PE.
  • The absolute number of CMV antigen-specific CD8+ T cells is determined by using the absolute count of CD3+CD8+ events calculated from data collected in panel 1 and the number of MHC tetramer-positive events expressed as percentage of CD3+CD8+ events determined in panel 2 using the following equations:
  • Panel 1

  • (CD3+CD8+ events×concentrationen of flow count beads in sample)/flow count events=absolute count CD3+CD8+ cells/volume blood
  • Panel 2

  • (absolute count CD3+CD8+ cells)×(MHC tetramer-positive events as percentage of CD8+ cells/100)=CD3+CD8+ MHC tetramer+cells/volume blood
  • Detection and Enumeration of CMV Antigen-Specific CD8-Positive T Lymphocytes in Whole Blood by Flow Cytometry.
  • Reagent Preparation
    • 1. Lyse/Fixative solution: Calculate the total volume of Lyse/Fixative solution required (panel 1—1 mL/tube, panel 2—2 mL/tube). Add 25 μL of iTAg™ MHC Dextramer Fixative Reagent to 1 mL of iTAg MHC Dextramer Lyse Reagent.
    • 2. 0.1% formaldehyde in PBS: Calculate the total volume of 0.1% formaldehyde/PBS fixative solution required (panel 2 only—0.5 mL/tube). Add 12.5 μL iTAg MHC Dextramer Fixative Reagent to 1 mL of PBS.
    • 3. Bring all monoclonal antibody reagents, IMMUNO-TROL™ Control Cells and iTAg MHC Dextramers to room temperature (RT) before pipetting. Vortex before use.
    • 4. On the same day of data acquisition by flow cytometry, remove Flow-Count™ Fluorospheres from 4° C. storage. Bring to RT prior to use. Vortex for 10-12 seconds and avoid excessive mixing to minimize air bubble formation.
  • Panel 1. Determination of Absolute T Cell Counts—Lyse-no-Wash
    • 1. Appropriately label tubes for each patient being tested.
    • 2. Pipette 10 μL of anti-CD3, 10 μL of anti-CD4, and 10 μL of anti-CD8 into the bottom of each tube.
    • 3. Pipette 100 μL of whole blood into the bottom of each tube.
    • 4. Vortex gently to ensure complete mixing of whole blood sample with antibody reagents.
    • 5. Incubate tubes at room temperature (18-25° C.) for 20-30 minutes, protected from light.
    • 6. Add 1 mL of Lyse/Fixative solution to each tube and vortex immediately for one second after each addition.
    • 7. Incubate at room temperature for at least 10 minutes, protected from light.
    • 8. Store prepared samples at 4° C., protected from light, until the addition of Flow-Count Fluorospheres within 24 hours.
    • 9. Pipette 100 μL of adequately mixed room-temperature Flow-Count Fluorospheres into each tube immediately prior to analysis by flow cytometry.
    • 10. Vortex each tube for 5 seconds to ensure proper mixing and resuspension of cells and fluorospheres.
    • 11. Samples must be analyzed within one hour. Repeat vortexing immediately prior to flow-cytometric acquisition.
  • Panel 2. Determination of Relative Percent of CMV Antigen-Specific CD8+T Cells—Lyse-with-Wash
    • 1. Appropriately label tubes for each patient being tested.
    • 2. Add 10 μL of specific iTAg MHC Class I Dextramer or Negative Dextramer and 10 μL each of anti-CD3 and anti-CD8 monoclonal antibody reagents into each tube.
    • 3. Add 200 μL of whole blood into each tube. Specimens with low leukocyte counts (<3.0 ×103/μL) or low lymphocyte counts (<0.5 ×103/μL) may require whole blood volumes up to 400 μL. Under these circumstances, up to 4 mL of Lyse/Fixative solution is required—all other reagent volumes remain as described.
    • 4. Vortex gently.
    • 5. Incubate at room temperature (18-25° C.) for 20-30 minutes, protected from light.
    • 6. Add 2 mL of Lyse/Fixative solution to each tube and vortex immediately for one second after each addition.
    • 7. Incubate at room temperature for at least 10 minutes, protected from light.
    • 8. Centrifuge tubes at 150×g for 5 minutes.
    • 9. Aspirate or decant the supernatant.
    • 10. Add 3 mL of PBS.
    • 11. Centrifuge tubes at 150×g for 5 minutes.
    • 12. Aspirate or decant the supernatant.
    • 13. Resuspend the cell pellet in 500 μL of PBS with 0.1% formaldehyde.
    • 14. Vortex each tube for 5 seconds.
    • 15. Store prepared samples at 4° C. protected from light for a minimum of 1 hour (maximum 24 hours) until analysis by flow cytometry.
    Example **Immune Monitoring 3
  • Any MHC multimer may be used to monitor the immune status of a patient following transplantation, thereby guiding the immune suppressive treatment. The procedure for the use of MHC dextramers to monitor immune status of a patient following transplantation described elsewhere herein may be followed replacing MHC dextramers with MHC dextramers.

Claims (2)

1. A method for detecting and/or analysing and/or partitioning one or more entities present in a sample, said method comprising the steps of
A) providing a sample by extracting the sample from a sample source,
B) preparing the sample for assaying by contacting said sample with one or more of i) a marker molecule specific for the one or more entities; ii) a labelling molecule specific for the one or more entities; and iii) a detection molecule comprising a marker molecule and a labelling molecule specific for the one or more entities, and
C) assaying the sample at least for the presence of the one or more entities, and optionally one or more further steps selected from
D) data processing and/or sample processing, such as sample analysis and/or partitioning of analysed entities, and
E) data interpretation and/or
F) further sample manipulations, such as cultivation and/or expansion of partitioned and/or isolated cells.
2-194. (canceled)
US12/647,747 2007-07-03 2009-12-28 Compiled Methods for Analysing and Sorting Samples Abandoned US20100248257A1 (en)

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Cited By (16)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20070202536A1 (en) * 2001-10-11 2007-08-30 Yamanishi Douglas T Methods and compositions for separating rare cells from fluid samples
US20130300865A1 (en) * 2012-05-08 2013-11-14 University Of Calcutta Static magnetic field induced differential fluorescence emission
US8838394B2 (en) 2012-02-03 2014-09-16 California Institute Of Technology Signal encoding and decoding in multiplexed biochemical assays
US20140291550A1 (en) * 2013-04-01 2014-10-02 National Institute Of Standards And Technology Flow cytometer systems and associated methods
US20150056649A1 (en) * 2012-01-13 2015-02-26 Konica Minolta, Inc. Method for quantifying cell of interest in blood, and method for evaluating system for quantifying said cell
US8969021B2 (en) 2001-10-11 2015-03-03 Aviva Biosciences Corporation Methods and compositions for detecting non-hematopoietic cells from a blood sample
US8986945B2 (en) 2006-07-14 2015-03-24 Aviva Biosciences Corporation Methods and compositions for detecting rare cells from a biological sample
US9920123B2 (en) 2008-12-09 2018-03-20 Genentech, Inc. Anti-PD-L1 antibodies, compositions and articles of manufacture
US10066263B2 (en) 2016-06-17 2018-09-04 California Institute Of Technology Nucleic acid reactions and related methods and compositions
US10436778B2 (en) * 2013-03-15 2019-10-08 Plexbio Co., Ltd. Methods and systems for multiplex assays
US10839509B2 (en) 2015-07-10 2020-11-17 3Scan Inc. Spatial multiplexing of histological stains
US10859910B2 (en) 2015-06-11 2020-12-08 Plexbio Co., Ltd. Image differentiated multiplex assays
US11796535B2 (en) 2016-09-16 2023-10-24 Plexbio Co., Ltd. Methods and systems for multiplex assays
US11866465B2 (en) 2017-04-27 2024-01-09 Juno Therapeutics Gmbh Oligomeric particle reagents and methods of use thereof
US11913024B2 (en) 2015-10-22 2024-02-27 Juno Therapeutics Gmbh Methods for culturing cells and kits and apparatus for same
US11959856B2 (en) 2017-09-11 2024-04-16 California Institute Of Technology Multiplexing and quantification in PCR with reduced hardware and requirements

Families Citing this family (70)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1536021A1 (en) * 2003-11-27 2005-06-01 Consortium National de Recherche en Genomique (CNRG) Method for HLA typing
US9090673B2 (en) 2003-12-12 2015-07-28 City Of Hope Synthetic conjugate of CpG DNA and T-help/CTL peptide
JP2009531324A (en) 2006-03-20 2009-09-03 ザ リージェンツ オブ ザ ユニバーシティ オブ カリフォルニア Engineered anti-prostatic stem cell antigen (PSCA) antibody for cancer targeting
EP2361930A3 (en) * 2007-03-26 2011-10-26 Dako Denmark A/S Multimers of MHC-peptide complexes and uses thereof in Borrelia infectious diseases
WO2009003493A2 (en) 2007-07-03 2009-01-08 Dako Denmark A/S Mhc multimers, methods for their generation, labeling and use
WO2009032949A2 (en) 2007-09-04 2009-03-12 The Regents Of The University Of California High affinity anti-prostate stem cell antigen (psca) antibodies for cancer targeting and detection
WO2009039854A2 (en) 2007-09-27 2009-04-02 Dako Denmark A/S Mhc multimers in tuberculosis diagnostics, vaccine and therapeutics
US10968269B1 (en) 2008-02-28 2021-04-06 Agilent Technologies, Inc. MHC multimers in borrelia diagnostics and disease
WO2009106073A2 (en) * 2008-02-28 2009-09-03 Dako Denmark A/S Mhc multimers in borrelia diagnostics and disease
WO2010009735A2 (en) 2008-07-23 2010-01-28 Dako Denmark A/S Combinatorial analysis and repair
GB0817244D0 (en) 2008-09-20 2008-10-29 Univ Cardiff Use of a protein kinase inhibitor to detect immune cells, such as T cells
WO2010037397A1 (en) * 2008-10-01 2010-04-08 Dako Denmark A/S Mhc multimers in cmv immune monitoring
EP2337795A2 (en) * 2008-10-01 2011-06-29 Dako Denmark A/S Mhc multimers in cancer vaccines and immune monitoring
US10369204B2 (en) 2008-10-02 2019-08-06 Dako Denmark A/S Molecular vaccines for infectious disease
US8246565B2 (en) 2009-02-25 2012-08-21 The Invention Science Fund I, Llc Device for passively removing a target component from blood or lymph of a vertebrate subject
US8317737B2 (en) * 2009-02-25 2012-11-27 The Invention Science Fund I, Llc Device for actively removing a target component from blood or lymph of a vertebrate subject
WO2011114346A1 (en) 2010-03-18 2011-09-22 Chetan Balar Chitin and related compounds for use in treating bacterial and viral infections
WO2011128893A2 (en) 2010-04-15 2011-10-20 Leukodx Ltd. Device, system and method for rapid determination of a medical condition
WO2011143075A2 (en) * 2010-05-08 2011-11-17 Veridex, Llc A simple and affordable method for immuophenotyping using a microfluidic chip sample preparation with image cytometry
EP2404618A1 (en) 2010-07-07 2012-01-11 Stichting Katholieke Universiteit meer in het bijzonder Radboud Universiteit Nijmegen Immunomodulatory protein constructs with a helical polymeric backbone.
US20120020938A1 (en) * 2010-07-26 2012-01-26 Searete Llc, A Limited Liability Corporation Of The State Of Delaware MHC- less cells
US9511151B2 (en) 2010-11-12 2016-12-06 Uti Limited Partnership Compositions and methods for the prevention and treatment of cancer
WO2012139110A2 (en) 2011-04-08 2012-10-11 Prognosys Biosciences, Inc. Peptide constructs and assay systems
US10988516B2 (en) 2012-03-26 2021-04-27 Uti Limited Partnership Methods and compositions for treating inflammation
EP3828278A1 (en) 2012-06-27 2021-06-02 Rutgers, the State University of New Jersey Rapid assays for t-cell activation by rna measurements using flow cytometry
US9603948B2 (en) 2012-10-11 2017-03-28 Uti Limited Partnership Methods and compositions for treating multiple sclerosis and related disorders
AU2013359469B2 (en) * 2012-12-10 2019-05-16 Seattle Biomedical Research Institute Cell-free biofragment compositions and related systems, devices, and methods
US10610861B2 (en) 2012-12-17 2020-04-07 Accellix Ltd. Systems, compositions and methods for detecting a biological condition
WO2014097287A1 (en) 2012-12-17 2014-06-26 Leukodx, Ltd. Systems and methods for detecting a biological condition
JP6464140B2 (en) 2013-03-13 2019-02-06 ヘルス リサーチ インコーポレイテッドＨｅａｌｔｈ Ｒｅｓｅａｒｃｈ， Ｉｎｃ． Compositions and methods for using recombinant T cell receptors to directly recognize tumor antigens
US11231419B2 (en) * 2013-03-15 2022-01-25 Prognosys Biosciences, Inc. Methods for detecting peptide/MHC/TCR binding
CA2912365C (en) * 2013-05-14 2021-12-07 Oslo Universitetssykehus Hf Detection of gluten-specific t-cells
SG11201603487YA (en) 2013-11-04 2016-05-30 Uti Limited Partnership Methods and compositions for sustained immunotherapy
US20160033480A1 (en) * 2013-12-16 2016-02-04 Amy Huimeei Lo Immunovir and Components, Immunovir A, B, C, D Utility and Useful Processes
US9549914B2 (en) 2014-04-03 2017-01-24 The Johns Hopkins University Treatment of human cytomegalovirus by modulating Wnt
US11274278B2 (en) 2014-04-16 2022-03-15 Juno Therapeutics Gmbh Methods, kits and apparatus for expanding a population of cells
MA39867A (en) * 2014-04-23 2017-03-01 Juno Therapeutics Inc Methods for isolating, culturing, and genetically engineering immune cell populations for adoptive therapy
JP6956632B2 (en) * 2014-06-06 2021-11-02 ヘアレウ ホスピタル Judgment of antigen recognition by bar code labeling of MHC multimers
EP3155426B1 (en) 2014-06-13 2023-07-19 Immudex ApS General detection and isolation of specific cells by binding of labeled molecules
MX2017001650A (en) 2014-08-04 2017-04-27 Oncotherapy Science Inc Cdca1-derived peptide and vaccine containing same.
KR20220165831A (en) 2014-08-04 2022-12-15 온코세라피 사이언스 가부시키가이샤 Urlc10-derived peptide and vaccine containing same
EP3590954A3 (en) 2014-08-04 2020-03-25 OncoTherapy Science, Inc. Koc1-derived peptide and vaccine including same
DE102014113688A1 (en) * 2014-09-22 2016-03-24 Jacobs University Bremen Ggmbh ANTIGEN-SPECIFIC IMMUNE COLORING OF T CELLS
CN107847582A (en) 2015-05-06 2018-03-27 优迪有限合伙公司 Nanoparticulate compositions for perennial treatment
CN107636162B (en) 2015-06-01 2021-12-10 加利福尼亚技术学院 Compositions and methods for screening T cells with antigens directed to specific populations
WO2017031216A1 (en) * 2015-08-19 2017-02-23 The Johns Hopkins University Compositions and methods for diagnosing and treating lyme disease
EP3360886B1 (en) 2015-10-08 2023-01-25 Oncotherapy Science, Inc. Foxm1-derived peptide, and vaccine including same
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AU2016341527B2 (en) 2015-10-22 2023-04-27 Juno Therapeutics Gmbh Methods, kits, agents and apparatuses for transduction
US10371610B2 (en) 2016-02-23 2019-08-06 Noul Co., Ltd. Contact-type patch, staining method using the same, and manufacturing method thereof
KR20170099737A (en) 2016-02-23 2017-09-01 노을 주식회사 Contact-type staining patch and staining method using the same
JP2019521080A (en) * 2016-04-27 2019-07-25 ユニバーシティ・ヘルス・ネットワーク Peptide-HLA complex and method for producing the same
WO2018115146A1 (en) 2016-12-21 2018-06-28 Danmarks Tekniske Universitet Antigen presenting scaffolds for immune-cell manipulation
MA49979A (en) 2017-08-09 2020-06-17 Juno Therapeutics Inc PROCESSES FOR THE PRODUCTION OF GENETICALLY MODIFIED CELL COMPOSITIONS AND RELATED COMPOSITIONS
EP3678699A1 (en) 2017-09-07 2020-07-15 University Of Oslo Vaccine molecules
JP6962247B2 (en) * 2018-03-14 2021-11-05 Jsr株式会社 Semiconductor surface treatment composition and semiconductor surface treatment method
EP3810188A1 (en) 2018-06-20 2021-04-28 Danmarks Tekniske Universitet Scaffolds with stabilized mhc molecules for immune-cell manipulation
TW202023581A (en) 2018-08-02 2020-07-01 日商腫瘤療法 科學股份有限公司 Cdca1-derived peptide and vaccine containing same
DE102018122546B3 (en) * 2018-09-14 2019-12-05 Immatics Biotechnologies Gmbh High-throughput peptide MHC affinity screening method for TCR ligands
WO2020081537A1 (en) * 2018-10-16 2020-04-23 Texas Tech University System Method to express, purify, and biotinylate eukaryotic cell-derived major histocompatibility complexes
CN109321524A (en) * 2018-11-05 2019-02-12 中国医学科学院血液病医院（血液学研究所） The cellifugal method of one kind point
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KR20220044687A (en) * 2019-08-09 2022-04-11 프라운호퍼 게젤샤프트 쭈르 푀르데룽 데어 안겐반텐 포르슝 에. 베. Production of the MHC II/CII complex
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US20230265157A1 (en) * 2020-06-24 2023-08-24 Repertoire Immune Medicines, Inc. Mhc multimer expression constructs and uses thereof
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Citations (80)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4336173A (en) * 1978-02-21 1982-06-22 Sintef Process for preparing an aqueous emulsion or dispersion of a partly water-soluble material, and optionally further conversion of the prepared dispersion or emulsion to a polymer dispersion when the partly water-soluble material is a polymerizable monomer
US4387164A (en) * 1980-11-05 1983-06-07 Fmc Corporation Method and apparatus for chemical analysis using reactive reagents dispersed in soluble film
US4654267A (en) * 1982-04-23 1987-03-31 Sintef Magnetic polymer particles and process for the preparation thereof
US4876190A (en) * 1987-10-21 1989-10-24 Becton Dickinson & Company Peridinin-chlorophyll complex as fluorescent label
US5039487A (en) * 1987-12-22 1991-08-13 Board Of Regents, The University Of Texas System Methods for quantifying components in liquid samples
US5130297A (en) * 1988-06-23 1992-07-14 Anergen, Inc. Conjugates useful in ameliorating autoimmunity MHC-II-peptide
US5194425A (en) * 1988-06-23 1993-03-16 Anergen, Inc. Mhc-mediated toxic conjugates useful in ameliorating autoimmunity
US5312744A (en) * 1992-04-30 1994-05-17 Olympus Optical Co., Ltd. Method of immobilizing and preserving an immunologically reactive substance
US5627037A (en) * 1990-08-07 1997-05-06 Becton Dickinson And Company One step method for detection and enumeration of absolute counts of one more cell populations in a sample
US5635363A (en) * 1995-02-28 1997-06-03 The Board Of Trustees Of The Leland Stanford Junior University Compositions and methods for the detection, quantitation and purification of antigen-specific T cells
US5652342A (en) * 1988-06-14 1997-07-29 Cel-Sci Corporation Heterofunctional cellular immunological reagents, vaccines containing same and methods for the use of same
US5807552A (en) * 1995-08-04 1998-09-15 Board Of Regents, The University Of Texas System Compositions for conferring immunogenicity to a substance and uses thereof
US5869270A (en) * 1996-01-31 1999-02-09 Sunol Molecular Corporation Single chain MHC complexes and uses thereof
US5891741A (en) * 1997-05-16 1999-04-06 Coulter International Corp. Antibody-aminodextran-phycobiliprotein conjugates
US5955079A (en) * 1992-02-11 1999-09-21 Henry Jackson Foundation For The Advancement Of Military Medicine Dual carrier immunogenic construct
US6015884A (en) * 1996-03-28 2000-01-18 The Johns Hopkins University Soluble divalent and multivalent heterodimeric analogs of proteins
US6074645A (en) * 1996-11-12 2000-06-13 City Of Hope Immuno-reactive peptide CTL epitopes of human cytomegalovirus
US6090587A (en) * 1993-10-25 2000-07-18 Corixa Corporation Prokaryotic expression of MHC proteins
US6106840A (en) * 1988-06-23 2000-08-22 Anergen, Inc. MHC conjugates useful in ameliorating autoimmunity
US6129916A (en) * 1991-04-19 2000-10-10 Tanox, Inc. Method of Increasing activation on proliferation of T cells using antibody-microbead conjugates
US6140113A (en) * 1996-03-28 2000-10-31 The Johns Hopkins University Polynucleotides encoding molecular complexes which modify immune responses
US6197928B1 (en) * 1997-03-14 2001-03-06 The Regents Of The University Of California Fluorescent protein sensors for detection of analytes
US6197302B1 (en) * 1996-07-18 2001-03-06 Children's Hospital Medical Center Method of stimulating T cells
US6232445B1 (en) * 1997-10-29 2001-05-15 Sunol Molecular Corporation Soluble MHC complexes and methods of use thereof
US6248564B1 (en) * 1997-08-29 2001-06-19 Harvard University Mutant MHC class I molecules
US6268411B1 (en) * 1997-09-11 2001-07-31 The Johns Hopkins University Use of multivalent chimeric peptide-loaded, MHC/ig molecules to detect, activate or suppress antigen-specific T cell-dependent immune responses
US6306605B1 (en) * 1998-12-03 2001-10-23 Sunol Molecular Corporation Methods for making recombinant cells
US6335173B1 (en) * 1999-01-12 2002-01-01 Verve, Ltd. C/O James Bell Methods for detecting an analyte of interest using tyramide coating technology
US6387622B1 (en) * 1997-05-16 2002-05-14 Coulter International Corp. Ligand-aminodextran-marker conjugates and uses thereof
US20020058787A1 (en) * 1996-08-05 2002-05-16 Jack L. Strominger Mhc binding peptide oligomers and methods of use
US20020082411A1 (en) * 2001-01-23 2002-06-27 Darrick Carter Immune mediators and related methods
US20020119149A1 (en) * 1998-05-19 2002-08-29 Jakobsen Bent Karsten Multivalent T cell receptor complexes
US6451769B1 (en) * 1996-06-14 2002-09-17 Pasteur Merieux Serums Et Vaccins Compositions and methods for administering Borrelia DNA
US6458933B1 (en) * 1998-05-20 2002-10-01 Immunomedics, Inc. Therapeutic using a bispecific antibody
US6458354B1 (en) * 1996-03-28 2002-10-01 The Johns Hopkins University Molecular complexes which modify immune responses
US20030017447A1 (en) * 2000-03-17 2003-01-23 Barnardo Martin C Method
US6534633B1 (en) * 1998-10-21 2003-03-18 Altor Bioscience Corporation Polyspecific binding molecules and uses thereof
US6548067B1 (en) * 1988-07-28 2003-04-15 Gerhard Seeman Antigenic constructs of major histocompatibility complex class I antigens with specific carrier molecules, the preparation and use thereof
US20030073102A1 (en) * 2001-04-05 2003-04-17 Virginia Mason Research Center Methods of MHC class II epitope mapping, detection of autoimmune T cells and antigens, and autoimmune treatment
US20030096432A1 (en) * 1999-09-21 2003-05-22 Avidex Limited Screening methods
US20030104635A1 (en) * 1999-09-21 2003-06-05 Avidex Limited Screening methods
US20030171290A1 (en) * 2000-08-03 2003-09-11 Carr Francis J. Peptides presented by cells
US20030199438A1 (en) * 2001-04-09 2003-10-23 Shaw Allan Christian Method for identification of proteins from intracellular bacteria
US20040068100A1 (en) * 2001-05-24 2004-04-08 Jean-Pierre Mach Multicomponent conjugates which bind to target molecules and stimulate cell lysis
US20040072262A1 (en) * 2002-10-11 2004-04-15 Montero-Julian Felix A. Methods and systems for detecting MHC class I binding peptides
US20040082012A1 (en) * 2000-12-28 2004-04-29 Busch Dirk H. Reversible mhc multimer staining for functional purification of antigen-specific t cells
US20040086520A1 (en) * 1999-10-20 2004-05-06 City Of Hope CTL epitope analogs
US20040137642A1 (en) * 2001-04-10 2004-07-15 Volker Erfle MHC tetramers
US20040143094A1 (en) * 2003-02-10 2004-07-22 Alena Donda Multicomponent conjugates which bind to target molecules and stimulate T cell lysis
US20040141958A1 (en) * 1998-10-05 2004-07-22 M&E Biotech A/S Novel methods for therapeutic vaccination
US20040209314A1 (en) * 1999-09-06 2004-10-21 Institut National De La Sante Et De La Recherche Medicale France Means for detection and purification of CD8+ T lymphocyte populations specific to peptides presented in the context of HLA
US20040209295A1 (en) * 2002-08-21 2004-10-21 Nikolai Franz Gregor Schwabe Chimeric MHC protein and oligomer thereof
US20050003431A1 (en) * 1996-08-16 2005-01-06 Wucherpfennig Kai W. Monovalent, multivalent, and multimeric MHC binding domain fusion proteins and conjugates, and uses therefor
US20050074822A1 (en) * 2003-07-29 2005-04-07 Douglas Nixon Method of detecting antigen-specific T lymphocytes
US20050074848A1 (en) * 2003-10-06 2005-04-07 Schwabe Nikolai Franz Gregor Chimeric MHC protein and oligomer thereof for specific targeting
US20050079152A1 (en) * 2003-06-17 2005-04-14 Bot Adrian Ian Methods of elicit, enhance and sustain immune responses against MHC class I-restricted epitopes, for prophylactic or therapeutic purposes
US20050095655A1 (en) * 2003-11-03 2005-05-05 Montero-Julian Felix A. Solution-based methods for detecting MHC-binding peptides
US20050208529A1 (en) * 2003-12-01 2005-09-22 Lars Winther Methods and compositions for immuno-histochemical detection
US20050214284A1 (en) * 2003-11-10 2005-09-29 Altor Bioscience Corporation Soluble TCR molecules and methods of use
US20060018929A1 (en) * 2000-03-23 2006-01-26 City Of Hope Protein kinase deficient, immunologically active CMVpp65 mutants
US20060018878A1 (en) * 2003-03-11 2006-01-26 City Of Hope Dual antigen specific T cells with trafficking ability
US20060084116A1 (en) * 2004-09-13 2006-04-20 Xencor, Inc. Analysis of MHC-peptide binding interactions
US7041442B1 (en) * 1999-06-04 2006-05-09 Florian Kern Peptides for vaccinating against human CMV
US7064190B1 (en) * 1994-01-20 2006-06-20 Roche Diagnostics Gmbh Antigen-specific, activated T lymphocytes, detection and use
US20060141540A1 (en) * 1992-10-21 2006-06-29 Stefan Miltenyi Direct selection of cells by secretion product
US7074904B2 (en) * 1994-07-29 2006-07-11 Altor Bioscience Corporation MHC complexes and uses thereof
US20060166214A1 (en) * 2003-03-10 2006-07-27 Yukio Kato Marker for detecting mesenchymal stem cell and method of distinguishing mesenchymal stem cell using the marker
US20060166875A1 (en) * 2002-10-09 2006-07-27 Avidex Limited Single chain recombinant t cell receptors
US7116407B2 (en) * 1998-12-15 2006-10-03 Union Biometrica, Inc. System for axial pattern analysis of multicellular organisms
US20070026503A1 (en) * 2005-07-22 2007-02-01 City Of Hope Polyomavirus cellular epitopes and uses therefor
US7202349B2 (en) * 2001-01-12 2007-04-10 Becton, Dickinson And Company Intrinsically fluorescent, self-multimerizing MHC fusion proteins and complexes thereof
US20070134814A1 (en) * 2005-12-09 2007-06-14 Kajander E O Methods and compositions for the detection of calcifying nano-particles, identification and quantification of associated proteins thereon, and correlation to disease
US20070154953A1 (en) * 2003-03-05 2007-07-05 Herwig Brunner Identification of antigen epitopes
US20070178532A1 (en) * 2003-03-24 2007-08-02 The Gov.Of The Us As Represented By The Secretary Identification, quantification, and characterization of t cells and t cell antigens
US20080219947A1 (en) * 2002-04-19 2008-09-11 Linette Gerald P Single chain trimers and uses therefor
US20090004213A1 (en) * 2007-03-26 2009-01-01 Immatics Biotechnologies Gmbh Combination therapy using active immunotherapy
US7524503B2 (en) * 2001-06-26 2009-04-28 The Council Of The Queensland Institute Of Medical Research Human cytomegalovirus (HCMV) cytotoxic T cell epitopes, polyepitopes compositions comprising same and diagnostic and prophylactic and therapeutic uses therefor
US20100159594A1 (en) * 2006-04-04 2010-06-24 Washington University Single chain trimers and uses therefor
US20120020998A1 (en) * 2008-05-16 2012-01-26 Etablissement Francais Du Sang Plasmacytoid dendritic cell line used in active or adoptive cell therapy
US8114669B2 (en) * 2002-10-03 2012-02-14 Plasticell Limited Cell culture

Family Cites Families (133)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
DK590288D0 (en) 1988-10-24 1988-10-24 Symbicom Ab CHEMICAL COMPOUNDS
US4015908A (en) 1976-03-18 1977-04-05 Amsted Industries Incorporated Multiple-piece crankshaft
DE2805826A1 (en) 1978-02-11 1979-08-23 Roehm Gmbh IMPROVED Grafting Techniques
US4485045A (en) 1981-07-06 1984-11-27 Research Corporation Synthetic phosphatidyl cholines useful in forming liposomes
US4544545A (en) 1983-06-20 1985-10-01 Trustees University Of Massachusetts Liposomes containing modified cholesterol for organ targeting
US5260422A (en) 1988-06-23 1993-11-09 Anergen, Inc. MHC conjugates useful in ameliorating autoimmunity
US5468481A (en) 1988-06-23 1995-11-21 Amergen, Inc. MHC class II-peptide conjugates useful in ameliorating autoimmunity
DE4015911A1 (en) 1989-09-19 1991-03-28 Max Planck Gesellschaft VACCINE AGAINST LYME DISEASE
DE59010422D1 (en) 1989-12-22 1996-08-22 Mikrogen Molekularbiol Entw IMMUNOLOGICALLY ACTIVE PROTEINS FROM BORRELIA BURGDORFERI, RELATED TEST KITS AND VACCINE
GB9007966D0 (en) 1990-04-09 1990-06-06 Dynal As Antigen/anti-antigen cleavage
CA2084413A1 (en) 1990-06-15 1991-12-16 Richard A. Flavell Compositions and methods for the prevention and diagnosis of lyme disease
WO1994011078A1 (en) 1992-11-16 1994-05-26 Immunivest Corporation Magnetic immobilization and manipulation of biological entities
AU8900691A (en) 1990-11-19 1992-06-11 Queen's University At Kingston Hiv marker/aids vaccine
CA2065658A1 (en) 1991-04-19 1992-10-20 Tse-Wen Chang Conjugates of liposomes or microbeads and antibodies specific for t lymphocytes and their use in vivo immune modulators
DE69232875T2 (en) 1991-04-23 2003-08-21 Anergen Inc MHC conjugates to improve autoimmunity.
US6228982B1 (en) 1992-05-22 2001-05-08 Benget Norden Double-stranded peptide nucleic acids
DK51092D0 (en) 1991-05-24 1992-04-15 Ole Buchardt OLIGONUCLEOTIDE ANALOGUE DESCRIBED BY PEN, MONOMERIC SYNTHONES AND PROCEDURES FOR PREPARING THEREOF, AND APPLICATIONS THEREOF
US5714331A (en) 1991-05-24 1998-02-03 Buchardt, Deceased; Ole Peptide nucleic acids having enhanced binding affinity, sequence specificity and solubility
US5641625A (en) 1992-05-22 1997-06-24 Isis Pharmaceuticals, Inc. Cleaving double-stranded DNA with peptide nucleic acids
US5719262A (en) 1993-11-22 1998-02-17 Buchardt, Deceased; Ole Peptide nucleic acids having amino acid side chains
US5766855A (en) 1991-05-24 1998-06-16 Buchardt, Deceased; Ole Peptide nucleic acids having enhanced binding affinity and sequence specificity
US5539082A (en) 1993-04-26 1996-07-23 Nielsen; Peter E. Peptide nucleic acids
WO1992021972A1 (en) 1991-06-06 1992-12-10 Immulogic Pharmaceutical Corporation Rapid screening assay for the identification of ligand-receptor interactions
CA2112992C (en) 1991-07-04 2002-06-11 Allan O. F. Lihme Water-soluble, polymer-based reagents and conjugates comprising moieties derived from divinyl sulfone
US6872550B1 (en) 1991-07-11 2005-03-29 Baxter Vaccine Ag Immunogenic formulation of OspC antigen vaccines for the prevention and treatment of lyme disease and recombinant methods for the preparation of such antigens
EP0878545A3 (en) 1991-08-15 1998-12-16 SMITHKLINE BEECHAM BIOLOGICALS s.a. OSP a protein of borrelia burgdorferi subgroups, encoding genes and vaccines
ATE234365T1 (en) 1991-10-22 2003-03-15 Symbicom Ab IMPROVEMENTS IN DIAGNOSIS AND PROPHYLAXIS OF BORRELIA BURGDORFERI
AU3220593A (en) 1991-11-19 1993-06-15 Anergen, Inc. Soluble mhc molecules and their uses
US5583031A (en) 1992-02-06 1996-12-10 President And Fellows Of Harvard College Empty major histocompatibility class II heterodimers
GB9211979D0 (en) 1992-06-05 1992-07-15 Buchard Ole Uses of nucleic acid analogues
ES2182832T3 (en) 1992-10-15 2003-03-16 Toray Industries PROCEDURE FOR THE PRODUCTION OF A CHMII ANTIGEN RECOMBINANT PROTEIN IN MICROORGANISMS.
AU5729294A (en) 1992-11-25 1994-06-22 Tanox Biosystems, Inc. Conjugates and constructs including anti-cd28 and anti-cd3 binding molecules
US5527675A (en) 1993-08-20 1996-06-18 Millipore Corporation Method for degradation and sequencing of polymers which sequentially eliminate terminal residues
DE4331012A1 (en) 1993-09-13 1995-03-16 Bayer Ag Nucleic acid-binding oligomers with N-branching for therapy and diagnostics
AU8091694A (en) 1993-10-26 1995-05-22 United Biomedical Inc. Structured synthetic antigen libraries as diagnostics, vaccines and therapeutics
WO2002016422A2 (en) 2000-08-18 2002-02-28 Research Foundation Of The State University Of New York Recombinant constructs of borrelia burgdorferi
ES2219657T3 (en) 1993-11-01 2004-12-01 Brookhaven Science Associates CHEMICAL PROTEINS THAT INCLUDE BORRELIA POLYPEPTIDES: USES FOR THE SAME.
US5620862A (en) 1993-11-24 1997-04-15 University Of Connecticut Methods for diagnosing early Lyme disease
GB2284209A (en) 1993-11-25 1995-05-31 Ole Buchardt Nucleic acid analogue-induced transcription of RNA from a double-stranded DNA template
EP0776339B2 (en) 1994-07-29 2010-01-20 Sunol Molecular Corporation Mhc complexes and uses thereof
US5705333A (en) 1994-08-05 1998-01-06 The Regents Of The University Of California Peptide-based nucleic acid mimics(PENAMS)
US20030118594A1 (en) 1995-06-07 2003-06-26 Bishwajit Nag Stable formulations of mhc-peptide complexes
WO1997005239A1 (en) 1995-07-25 1997-02-13 Celltherapy, Inc. Autologous immune cell therapy: cell compositions, methods and applications to treatment of human disease
US6001964A (en) * 1995-09-20 1999-12-14 Albert Einstein College Of Medicine Of Yeshiva University Peptides which bind to anti-double stranded DNA antibody
ATE232212T1 (en) 1996-05-02 2003-02-15 Dako As USE OF PEPTIDE FRAGMENTS DERIVED FROM OSP-C FOR DIAGNOSTIC METHODS
AU3036597A (en) 1996-05-21 1997-12-09 Institut National De La Sante Et De La Recherche Medicale (Inserm) Method for using peptide complexes/major histocompatibility complex to obtain or purify antigen-specific t cells and to stimulate t cells
FR2751989B1 (en) 1996-08-01 1998-10-30 Soc D Rech Et De Dev En Activa METHOD FOR IDENTIFYING EUKARYOTIC CELLS BY FIXING SAID CELLS ON A CARRIER AND CARRIER FOR CARRYING OUT SAID METHOD
DK0935607T3 (en) 1996-08-16 2004-12-06 Harvard College Soluble monovalent and multivalent MHC class II fusion proteins and their applications
US6562345B1 (en) 1996-11-12 2003-05-13 City Of Hope Immuno-reactive peptide CTL epitopes of human cytomegalovirus
US6156317A (en) 1996-11-12 2000-12-05 City Of Hope Immuno-reactive peptide CTL epitopes of human cytomegalovirus
CA2271784A1 (en) 1996-11-12 1998-05-22 City Of Hope Immuno-reactive peptide ctl epitopes of human cytomegalovirus
US5998204A (en) 1997-03-14 1999-12-07 The Regents Of The University Of California Fluorescent protein sensors for detection of analytes
US6107470A (en) 1997-05-29 2000-08-22 Nielsen; Peter E. Histidine-containing peptide nucleic acids
ATE325619T1 (en) 1997-07-10 2006-06-15 Mannkind Corp DEVICE FOR INDUCING A CTL RESPONSE
GB9718463D0 (en) 1997-08-29 1997-11-05 Dynal As Biomolecules
DE19740735A1 (en) 1997-09-16 1999-03-18 Max Planck Gesellschaft Antibodies specific for Borrelia burgdorferi 24 kD antigen OspC
CA2302779C (en) 1997-09-16 2010-02-02 Oregon Health Sciences University Recombinant mhc molecules useful for manipulation of antigen-specific t-cells
AU750173B2 (en) 1997-11-10 2002-07-11 Statens Serum Institut Nucleic acid fragments and polypeptide fragments derived from M. tuberculosis
EP1051619B1 (en) 1998-01-19 2006-08-09 Florian Kern Method for identifying t-cell stimulating protein fragments
IL137706A0 (en) 1998-02-19 2001-10-31 Harvard College Mhc fusion proteins and conjugates
WO1999050637A2 (en) 1998-03-27 1999-10-07 Ludwig Institute For Cancer Research Isolated multimeric complexes useful in analysis of t cells, peptides useful in making the complexes, and uses thereof
EP0949508A1 (en) 1998-04-08 1999-10-13 Dako A/S Method, antigen complex and kit for diagnosing Lyme borreliosis
GB9810099D0 (en) 1998-05-11 1998-07-08 Isis Innovation Novel molecule and diagnostic method
DK1100922T3 (en) 1998-07-31 2008-06-16 Gundersen Lutheran Medical Fou Applications of one or more borreliacid epitopes on outer membrane protein C from Borrelia burgdorferi (OspC) as a vaccine
CA2343313A1 (en) 1998-09-14 2000-03-23 Lars Ostergaard Pedersen A method of producing a functional immunoglobulin superfamily protein
WO2000021989A1 (en) 1998-10-09 2000-04-20 Medimmune, Inc. Decorin binding proteins dbp a and b and genes encoding them
WO2000023053A2 (en) 1998-10-20 2000-04-27 Salvatore Albani Artificial antigen-specific cells and related methods
PT1179176E (en) 1999-04-16 2006-07-31 Ortho Mcneil Pharm Inc METHOD FOR DETECTING T-CELLS SPECIFIC TO ANTIGEN
AU5751000A (en) 1999-06-18 2001-01-09 Brook Biotechnologies, Inc. Groups of borrelia burgdorferi and borrelia afzelii that cause lyme disease in humans
US6605711B1 (en) 1999-11-15 2003-08-12 Ludwig Institute For Cancer Research NY-ESO-1 peptide derivatives, and uses thereof
DE10009341A1 (en) 2000-02-22 2001-09-06 Florian Kern Method for antigen-specific stimulation of T lymphocytes
EP1307211A4 (en) 2000-03-22 2005-01-05 Corixa Corp Immune mediators and related methods
AU2001249444A1 (en) 2000-03-28 2001-10-08 The Government Of The United States Of America, As Represented By The Secretary Of The Department Of Health And Human Services Methods and compositions for the simultaneous detection of multiple analytes
AU2000243137A1 (en) 2000-04-20 2001-11-07 Salvatore Albani Methods for isolation, quantification, characterization and modulation of antigen-specific t cells
WO2001090198A1 (en) 2000-05-24 2001-11-29 Ludwig Institute For Cancer Research Multicomponent conjugates which bind to target molecules and stimulate cell lysis
JP2004501364A (en) 2000-05-25 2004-01-15 スノル・モレキュラー・コーポレーション Modulation of T-cell receptor interaction
US6517838B1 (en) 2000-06-16 2003-02-11 The Texas A&M University System Decorin binding protein essential peptides and methods of use
US7132109B1 (en) 2000-10-20 2006-11-07 University Of Connecticut Health Center Using heat shock proteins to increase immune response
AU2002240818C1 (en) * 2001-03-14 2008-11-06 Agilent Technologies, Inc. MHC molecule constructs and their uses for diagnosis and therapy
US6811785B2 (en) 2001-05-07 2004-11-02 Mount Sinai School Of Medicine Of New York University Multivalent MHC class II—peptide chimeras
US7902121B2 (en) 2001-07-02 2011-03-08 The Board Of Trustees Of The Leland Stanford Junior University MHC-antigen arrays for detection and characterization of immune responses
GB0120042D0 (en) 2001-08-16 2001-10-10 Avidex Ltd Methods
US20050221381A1 (en) 2002-02-28 2005-10-06 Christof Klade Method for isolating ligands
US20060234309A1 (en) 2002-05-08 2006-10-19 Northwest Biotherapeutics, Inc Quality assays for antigen presenting cells
US20030228258A1 (en) 2002-05-30 2003-12-11 David Scheinberg Suicide tetramers and uses thereof
AU2003277865B2 (en) 2002-06-25 2008-08-21 City Of Hope Adjuvant-free peptide vaccine
BR0313101A (en) 2002-08-12 2005-06-28 Queensland Inst Med Res Novel immunogenic lipopeptides comprising t-helper lymphocyte epitopes and cytotoxic t lymphocytes (ctl), and methods for their production and use
DE10247014A1 (en) 2002-10-09 2004-04-22 Erfle, Volker, Prof. Priv.-Doz. Dr. New multimer of major histocompatibility complex molecules, useful e.g. for sorting specific T cells for therapeutic application, includes a chromoprotein as multimerizing agent
US20040223977A1 (en) * 2003-02-03 2004-11-11 City Of Hope Fusion peptide HIV vaccines
US7163685B2 (en) 2003-04-16 2007-01-16 City Of Hope Human cytomegalovirus antigens expressed in MVA and methods of use
ES2327643T3 (en) 2003-07-11 2009-11-02 Alphavax, Inc. VACCINES AGAINST CITOMEGALOVIRUS BASED ON ALFAVIRUS.
GB0318096D0 (en) 2003-08-01 2003-09-03 Queen Mary & Westfield College Vaccine
RU2260047C2 (en) 2003-10-23 2005-09-10 Государственное учреждение Научно-исследовательский институт биохимии Сибирского отделения Российской академии медицинских наук Recombinant plasmid dna providing synthesis of borrelia garinii immunodominant protein for lyme-borreliosis diagnosis
KR101216655B1 (en) * 2003-11-19 2013-01-02 수르벡 에이피에스 PROTEINS BELONGING TO THE Bcl-2 FAMILY AND FRAGMENTS THEREOF, AND THEIR USE IN CANCER PATIENTS
WO2005111624A2 (en) * 2004-05-07 2005-11-24 Beckman Coulter, Inc. Mhc bridging system for detecting ctl-mediated lysis of antigen presenting cells
US20060073159A1 (en) 2004-05-25 2006-04-06 The Trustees Of The University Of Pennsylvania Human anti-cancer immunotherapy
DE102004026135A1 (en) 2004-05-25 2006-01-05 Immatics Biotechnologies Gmbh Tumor-associated peptides binding to MHC molecules
WO2006009838A2 (en) * 2004-06-17 2006-01-26 Beckman Coulter, Inc. Mycobacterium tuberculosis epitopes and methods of use thereof
WO2006014292A2 (en) 2004-07-02 2006-02-09 Dattwyler Raymond J Oral vaccine for borrelia
US20060228758A1 (en) 2004-09-13 2006-10-12 Xencor, Inc. Analysis of MHC-peptide binding interactions
US20060228759A1 (en) 2004-09-13 2006-10-12 Xencor, Inc. Analysis of MHC-peptide binding interactions
DE102004054545A1 (en) 2004-09-16 2006-04-06 Max-Delbrück-Centrum für Molekulare Medizin Change in the loading state of MHC molecules
WO2006056027A1 (en) 2004-11-29 2006-06-01 The Council Of The Queensland Institute Of Medical Research Human cytomegalovirus immunotherapy
AU2005321905A1 (en) 2004-12-29 2006-07-06 Mannkind Corporation Methods to bypass CD+4 cells in the induction of an immune response
GB2422834B8 (en) 2005-02-04 2007-01-04 Proimmune Ltd MHC oligomer and method of making the same
EP2329841A1 (en) * 2005-02-04 2011-06-08 Survac ApS Survivin peptide vaccine
US8541227B2 (en) 2005-02-25 2013-09-24 Dako Denmark A/S Cell counting
WO2006113622A2 (en) 2005-04-15 2006-10-26 University Of Maryland, Baltimore Direct vaccination of the bone marrow
US9017992B2 (en) 2005-07-01 2015-04-28 Dako Denmark A/S In situ hybridization detection method
US7794727B2 (en) 2005-11-29 2010-09-14 Virginia Commonwealth University Polyvalent chimeric OspC vaccinogen and diagnostic antigen
WO2007085266A1 (en) 2006-01-30 2007-08-02 Dako Denmark A/S High-speed quantification of antigen specific t-cells in whole blood by flow cytometry
BRPI0712716A2 (en) 2006-05-19 2012-05-22 Teva Pharma fusion protein, composition, nucleic acid construction, vector, transformed cell, isolated preparation of bacterially expressed inclusion bodies, process for producing a fusion protein, and methods for selectively killing a tumor cell and for treating a tumor cell expressing mesothelin on its surface
US20080171059A1 (en) 2006-08-07 2008-07-17 Shanshan Wu Howland Methods and compositions for increased priming of t-cells through cross-presentation of exogenous antigens
AU2007295927A1 (en) 2006-09-15 2008-03-20 Intercell Ag Borrelia antigens
EP2361930A3 (en) * 2007-03-26 2011-10-26 Dako Denmark A/S Multimers of MHC-peptide complexes and uses thereof in Borrelia infectious diseases
WO2009003493A2 (en) 2007-07-03 2009-01-08 Dako Denmark A/S Mhc multimers, methods for their generation, labeling and use
WO2009039854A2 (en) 2007-09-27 2009-04-02 Dako Denmark A/S Mhc multimers in tuberculosis diagnostics, vaccine and therapeutics
WO2009077173A2 (en) 2007-12-19 2009-06-25 Philochem Ag Dna-encoded chemical libraries
WO2009106073A2 (en) * 2008-02-28 2009-09-03 Dako Denmark A/S Mhc multimers in borrelia diagnostics and disease
WO2009114207A2 (en) 2008-03-14 2009-09-17 Sanofi Pasteur Biologics Co. Replication-defective flavivirus vaccines and vaccine vectors
CN102112626A (en) * 2008-04-09 2011-06-29 加州理工学院 Capture agents and related methods and systems for detecting and/or sorting targets
WO2009126816A1 (en) 2008-04-09 2009-10-15 Ventria Bioscience Production of ospa for lyme disease control
CA2758213C (en) 2008-04-10 2017-08-15 Thrombosis Research Institute Anti-atheroma vaccine
US20090324630A1 (en) * 2008-04-21 2009-12-31 Jensen Michael C Fusion multiviral chimeric antigen
CN102123732A (en) 2008-06-20 2011-07-13 杜克大学 Compositions, methods and kits for eliciting an immune response
WO2010009735A2 (en) 2008-07-23 2010-01-28 Dako Denmark A/S Combinatorial analysis and repair
GB0817244D0 (en) 2008-09-20 2008-10-29 Univ Cardiff Use of a protein kinase inhibitor to detect immune cells, such as T cells
WO2010037397A1 (en) 2008-10-01 2010-04-08 Dako Denmark A/S Mhc multimers in cmv immune monitoring
EP2337795A2 (en) 2008-10-01 2011-06-29 Dako Denmark A/S Mhc multimers in cancer vaccines and immune monitoring
US10369204B2 (en) 2008-10-02 2019-08-06 Dako Denmark A/S Molecular vaccines for infectious disease
US8735327B2 (en) 2010-01-07 2014-05-27 Jeansee, Llc Combinatorial DNA taggants and methods of preparation and use thereof
US10023657B2 (en) 2010-10-01 2018-07-17 Ludwig Institute For Cancer Research Ltd. Reversible protein multimers, methods for their production and use
EP3155426B1 (en) * 2014-06-13 2023-07-19 Immudex ApS General detection and isolation of specific cells by binding of labeled molecules

Patent Citations (100)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4336173A (en) * 1978-02-21 1982-06-22 Sintef Process for preparing an aqueous emulsion or dispersion of a partly water-soluble material, and optionally further conversion of the prepared dispersion or emulsion to a polymer dispersion when the partly water-soluble material is a polymerizable monomer
US4459378A (en) * 1978-02-21 1984-07-10 Sintef Monodisperse polymer particles and dispersions thereof
US4387164A (en) * 1980-11-05 1983-06-07 Fmc Corporation Method and apparatus for chemical analysis using reactive reagents dispersed in soluble film
US4654267A (en) * 1982-04-23 1987-03-31 Sintef Magnetic polymer particles and process for the preparation thereof
US4876190A (en) * 1987-10-21 1989-10-24 Becton Dickinson & Company Peridinin-chlorophyll complex as fluorescent label
US5039487A (en) * 1987-12-22 1991-08-13 Board Of Regents, The University Of Texas System Methods for quantifying components in liquid samples
US5652342A (en) * 1988-06-14 1997-07-29 Cel-Sci Corporation Heterofunctional cellular immunological reagents, vaccines containing same and methods for the use of same
US6096315A (en) * 1988-06-14 2000-08-01 Cel Sci Corporation Heterofunctional cellular immunological reagents, vaccines containing same and methods for the use of same
US5194425A (en) * 1988-06-23 1993-03-16 Anergen, Inc. Mhc-mediated toxic conjugates useful in ameliorating autoimmunity
US5284935A (en) * 1988-06-23 1994-02-08 Anergen, Inc. MHC-mediated toxic conjugates useful in ameliorating autoimmunity
US6451314B1 (en) * 1988-06-23 2002-09-17 Anergen, Inc. MHC conjugates useful in ameliorating autoimmunity
US5130297A (en) * 1988-06-23 1992-07-14 Anergen, Inc. Conjugates useful in ameliorating autoimmunity MHC-II-peptide
US6106840A (en) * 1988-06-23 2000-08-22 Anergen, Inc. MHC conjugates useful in ameliorating autoimmunity
US6548067B1 (en) * 1988-07-28 2003-04-15 Gerhard Seeman Antigenic constructs of major histocompatibility complex class I antigens with specific carrier molecules, the preparation and use thereof
US5627037A (en) * 1990-08-07 1997-05-06 Becton Dickinson And Company One step method for detection and enumeration of absolute counts of one more cell populations in a sample
US6129916A (en) * 1991-04-19 2000-10-10 Tanox, Inc. Method of Increasing activation on proliferation of T cells using antibody-microbead conjugates
US5955079A (en) * 1992-02-11 1999-09-21 Henry Jackson Foundation For The Advancement Of Military Medicine Dual carrier immunogenic construct
US5312744A (en) * 1992-04-30 1994-05-17 Olympus Optical Co., Ltd. Method of immobilizing and preserving an immunologically reactive substance
US20060141540A1 (en) * 1992-10-21 2006-06-29 Stefan Miltenyi Direct selection of cells by secretion product
US6090587A (en) * 1993-10-25 2000-07-18 Corixa Corporation Prokaryotic expression of MHC proteins
US7064190B1 (en) * 1994-01-20 2006-06-20 Roche Diagnostics Gmbh Antigen-specific, activated T lymphocytes, detection and use
US20060171954A1 (en) * 1994-01-20 2006-08-03 Roche Diagnostics Gmbh Antigen specific activated T-lymphocytes, detection and use
US7074904B2 (en) * 1994-07-29 2006-07-11 Altor Bioscience Corporation MHC complexes and uses thereof
US5635363A (en) * 1995-02-28 1997-06-03 The Board Of Trustees Of The Leland Stanford Junior University Compositions and methods for the detection, quantitation and purification of antigen-specific T cells
US5807552A (en) * 1995-08-04 1998-09-15 Board Of Regents, The University Of Texas System Compositions for conferring immunogenicity to a substance and uses thereof
US5869270A (en) * 1996-01-31 1999-02-09 Sunol Molecular Corporation Single chain MHC complexes and uses thereof
US20020034513A1 (en) * 1996-01-31 2002-03-21 Sunol Molecular Corporation MHC complexes and uses thereof
US6309645B1 (en) * 1996-01-31 2001-10-30 Sunol Molecular Corporation MHC molecules and uses thereof
US6015884A (en) * 1996-03-28 2000-01-18 The Johns Hopkins University Soluble divalent and multivalent heterodimeric analogs of proteins
US6140113A (en) * 1996-03-28 2000-10-31 The Johns Hopkins University Polynucleotides encoding molecular complexes which modify immune responses
US6458354B1 (en) * 1996-03-28 2002-10-01 The Johns Hopkins University Molecular complexes which modify immune responses
US20020127231A1 (en) * 1996-03-28 2002-09-12 Jonathan Schneck Soluble divalent and multivalent heterodimeric analogs of proteins
US6448071B1 (en) * 1996-03-28 2002-09-10 The Johns Hopkins University Soluble divalent and multivalent heterodimeric analogs of proteins
US6451769B1 (en) * 1996-06-14 2002-09-17 Pasteur Merieux Serums Et Vaccins Compositions and methods for administering Borrelia DNA
US6211342B1 (en) * 1996-07-18 2001-04-03 Children's Hospital Medical Center Multivalent MHC complex peptide fusion protein complex for stimulating specific T cell function
US6197302B1 (en) * 1996-07-18 2001-03-06 Children's Hospital Medical Center Method of stimulating T cells
US20020058787A1 (en) * 1996-08-05 2002-05-16 Jack L. Strominger Mhc binding peptide oligomers and methods of use
US20050003431A1 (en) * 1996-08-16 2005-01-06 Wucherpfennig Kai W. Monovalent, multivalent, and multimeric MHC binding domain fusion proteins and conjugates, and uses therefor
US6251399B1 (en) * 1996-11-12 2001-06-26 Hope City Immuno-reactive peptide CTL epitopes of human cytomegalovirus
US6074645A (en) * 1996-11-12 2000-06-13 City Of Hope Immuno-reactive peptide CTL epitopes of human cytomegalovirus
US20060112440A1 (en) * 1997-03-14 2006-05-25 The Regents Of The University Of California Fluorescent protein sensors for detection of analytes
US7060869B2 (en) * 1997-03-14 2006-06-13 The Regents Of The University Of California Fluorescent protein sensors for detection of analytes
US6197928B1 (en) * 1997-03-14 2001-03-06 The Regents Of The University Of California Fluorescent protein sensors for detection of analytes
US6734013B2 (en) * 1997-03-28 2004-05-11 The Johns Hopkins University Use of multivalent chimeric peptide-loaded, MHC/Ig molecules to detect, activate or suppress antigen-specific T cell-dependent immune responses
US20020006903A1 (en) * 1997-03-28 2002-01-17 Jonathan Schneck Use of multivalent chimeric peptide-loaded, MHC/Ig molecules to detect, activate or suppress antigen-specific T cell-dependent immune responses
US5891741A (en) * 1997-05-16 1999-04-06 Coulter International Corp. Antibody-aminodextran-phycobiliprotein conjugates
US6387622B1 (en) * 1997-05-16 2002-05-14 Coulter International Corp. Ligand-aminodextran-marker conjugates and uses thereof
US6248564B1 (en) * 1997-08-29 2001-06-19 Harvard University Mutant MHC class I molecules
US6268411B1 (en) * 1997-09-11 2001-07-31 The Johns Hopkins University Use of multivalent chimeric peptide-loaded, MHC/ig molecules to detect, activate or suppress antigen-specific T cell-dependent immune responses
US20040204565A1 (en) * 1997-09-11 2004-10-14 The Johns Hopkins University Use of multivalent chimeric peptide-loaded, MHC/IG molecules to detect, activate or suppress antigen-specific T cell-dependent immune responses
US20020091079A1 (en) * 1997-10-29 2002-07-11 Sunol Molecular Corporation Soluble MHC complexes and methods of use thereof
US6232445B1 (en) * 1997-10-29 2001-05-15 Sunol Molecular Corporation Soluble MHC complexes and methods of use thereof
US20060155115A1 (en) * 1998-05-19 2006-07-13 Avidex Limited Multivalent T cell receptor complexes
US20020119149A1 (en) * 1998-05-19 2002-08-29 Jakobsen Bent Karsten Multivalent T cell receptor complexes
US6458933B1 (en) * 1998-05-20 2002-10-01 Immunomedics, Inc. Therapeutic using a bispecific antibody
US20040141958A1 (en) * 1998-10-05 2004-07-22 M&E Biotech A/S Novel methods for therapeutic vaccination
US6534633B1 (en) * 1998-10-21 2003-03-18 Altor Bioscience Corporation Polyspecific binding molecules and uses thereof
US6306605B1 (en) * 1998-12-03 2001-10-23 Sunol Molecular Corporation Methods for making recombinant cells
US7116407B2 (en) * 1998-12-15 2006-10-03 Union Biometrica, Inc. System for axial pattern analysis of multicellular organisms
US6335173B1 (en) * 1999-01-12 2002-01-01 Verve, Ltd. C/O James Bell Methods for detecting an analyte of interest using tyramide coating technology
US7041442B1 (en) * 1999-06-04 2006-05-09 Florian Kern Peptides for vaccinating against human CMV
US20040209314A1 (en) * 1999-09-06 2004-10-21 Institut National De La Sante Et De La Recherche Medicale France Means for detection and purification of CD8+ T lymphocyte populations specific to peptides presented in the context of HLA
US20030096432A1 (en) * 1999-09-21 2003-05-22 Avidex Limited Screening methods
US20030104635A1 (en) * 1999-09-21 2003-06-05 Avidex Limited Screening methods
US20040086520A1 (en) * 1999-10-20 2004-05-06 City Of Hope CTL epitope analogs
US20030017447A1 (en) * 2000-03-17 2003-01-23 Barnardo Martin C Method
US20060018929A1 (en) * 2000-03-23 2006-01-26 City Of Hope Protein kinase deficient, immunologically active CMVpp65 mutants
US20030171290A1 (en) * 2000-08-03 2003-09-11 Carr Francis J. Peptides presented by cells
US20040082012A1 (en) * 2000-12-28 2004-04-29 Busch Dirk H. Reversible mhc multimer staining for functional purification of antigen-specific t cells
US7202349B2 (en) * 2001-01-12 2007-04-10 Becton, Dickinson And Company Intrinsically fluorescent, self-multimerizing MHC fusion proteins and complexes thereof
US20020082411A1 (en) * 2001-01-23 2002-06-27 Darrick Carter Immune mediators and related methods
US7094555B2 (en) * 2001-04-05 2006-08-22 Benaroya Research Institute At Virginia Mason Methods of MHC class II epitope mapping, detection of autoimmune T cells and antigens, and autoimmune treatment
US20030073102A1 (en) * 2001-04-05 2003-04-17 Virginia Mason Research Center Methods of MHC class II epitope mapping, detection of autoimmune T cells and antigens, and autoimmune treatment
US20030199438A1 (en) * 2001-04-09 2003-10-23 Shaw Allan Christian Method for identification of proteins from intracellular bacteria
US20040137642A1 (en) * 2001-04-10 2004-07-15 Volker Erfle MHC tetramers
US20040068100A1 (en) * 2001-05-24 2004-04-08 Jean-Pierre Mach Multicomponent conjugates which bind to target molecules and stimulate cell lysis
US7524503B2 (en) * 2001-06-26 2009-04-28 The Council Of The Queensland Institute Of Medical Research Human cytomegalovirus (HCMV) cytotoxic T cell epitopes, polyepitopes compositions comprising same and diagnostic and prophylactic and therapeutic uses therefor
US20080219947A1 (en) * 2002-04-19 2008-09-11 Linette Gerald P Single chain trimers and uses therefor
US20040209295A1 (en) * 2002-08-21 2004-10-21 Nikolai Franz Gregor Schwabe Chimeric MHC protein and oligomer thereof
US8114669B2 (en) * 2002-10-03 2012-02-14 Plasticell Limited Cell culture
US20060166875A1 (en) * 2002-10-09 2006-07-27 Avidex Limited Single chain recombinant t cell receptors
US20040072262A1 (en) * 2002-10-11 2004-04-15 Montero-Julian Felix A. Methods and systems for detecting MHC class I binding peptides
US20040143094A1 (en) * 2003-02-10 2004-07-22 Alena Donda Multicomponent conjugates which bind to target molecules and stimulate T cell lysis
US20070154953A1 (en) * 2003-03-05 2007-07-05 Herwig Brunner Identification of antigen epitopes
US20060166214A1 (en) * 2003-03-10 2006-07-27 Yukio Kato Marker for detecting mesenchymal stem cell and method of distinguishing mesenchymal stem cell using the marker
US20060018878A1 (en) * 2003-03-11 2006-01-26 City Of Hope Dual antigen specific T cells with trafficking ability
US20070178532A1 (en) * 2003-03-24 2007-08-02 The Gov.Of The Us As Represented By The Secretary Identification, quantification, and characterization of t cells and t cell antigens
US20050079152A1 (en) * 2003-06-17 2005-04-14 Bot Adrian Ian Methods of elicit, enhance and sustain immune responses against MHC class I-restricted epitopes, for prophylactic or therapeutic purposes
US20050074822A1 (en) * 2003-07-29 2005-04-07 Douglas Nixon Method of detecting antigen-specific T lymphocytes
US7364869B2 (en) * 2003-07-29 2008-04-29 The J. David Gladstone Institutes Method of detecting antigen-specific T lymphocytes
US20050074848A1 (en) * 2003-10-06 2005-04-07 Schwabe Nikolai Franz Gregor Chimeric MHC protein and oligomer thereof for specific targeting
US20050095655A1 (en) * 2003-11-03 2005-05-05 Montero-Julian Felix A. Solution-based methods for detecting MHC-binding peptides
US20050214284A1 (en) * 2003-11-10 2005-09-29 Altor Bioscience Corporation Soluble TCR molecules and methods of use
US20050208529A1 (en) * 2003-12-01 2005-09-22 Lars Winther Methods and compositions for immuno-histochemical detection
US20060084116A1 (en) * 2004-09-13 2006-04-20 Xencor, Inc. Analysis of MHC-peptide binding interactions
US20070026503A1 (en) * 2005-07-22 2007-02-01 City Of Hope Polyomavirus cellular epitopes and uses therefor
US20070134814A1 (en) * 2005-12-09 2007-06-14 Kajander E O Methods and compositions for the detection of calcifying nano-particles, identification and quantification of associated proteins thereon, and correlation to disease
US20100159594A1 (en) * 2006-04-04 2010-06-24 Washington University Single chain trimers and uses therefor
US20090004213A1 (en) * 2007-03-26 2009-01-01 Immatics Biotechnologies Gmbh Combination therapy using active immunotherapy
US20120020998A1 (en) * 2008-05-16 2012-01-26 Etablissement Francais Du Sang Plasmacytoid dendritic cell line used in active or adoptive cell therapy

Cited By (28)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20070202536A1 (en) * 2001-10-11 2007-08-30 Yamanishi Douglas T Methods and compositions for separating rare cells from fluid samples
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US8986945B2 (en) 2006-07-14 2015-03-24 Aviva Biosciences Corporation Methods and compositions for detecting rare cells from a biological sample
US9920123B2 (en) 2008-12-09 2018-03-20 Genentech, Inc. Anti-PD-L1 antibodies, compositions and articles of manufacture
US20150056649A1 (en) * 2012-01-13 2015-02-26 Konica Minolta, Inc. Method for quantifying cell of interest in blood, and method for evaluating system for quantifying said cell
EP2803998B1 (en) * 2012-01-13 2019-11-27 Konica Minolta, Inc. Method for evaluating system for quantifying cell of interest in blood
US8838394B2 (en) 2012-02-03 2014-09-16 California Institute Of Technology Signal encoding and decoding in multiplexed biochemical assays
US10068051B2 (en) 2012-02-03 2018-09-04 California Institute Of Technology Signal encoding and decoding in multiplexed biochemical assays
US11866768B2 (en) 2012-02-03 2024-01-09 California Institute Of Technology Signal encoding and decoding in multiplexed biochemical assays
US10770170B2 (en) 2012-02-03 2020-09-08 California Institute Of Technology Signal encoding and decoding in multiplexed biochemical assays
US11827921B2 (en) 2012-02-03 2023-11-28 California Institute Of Technology Signal encoding and decoding in multiplexed biochemical assays
US9459210B2 (en) * 2012-05-08 2016-10-04 University Of Calcutta Static magnetic field induced differential fluorescence emission
US20130300865A1 (en) * 2012-05-08 2013-11-14 University Of Calcutta Static magnetic field induced differential fluorescence emission
US10436778B2 (en) * 2013-03-15 2019-10-08 Plexbio Co., Ltd. Methods and systems for multiplex assays
US20140291550A1 (en) * 2013-04-01 2014-10-02 National Institute Of Standards And Technology Flow cytometer systems and associated methods
US11579522B2 (en) 2015-06-11 2023-02-14 Plexbio Co., Ltd. Image differentiated multiplex assays
US10859910B2 (en) 2015-06-11 2020-12-08 Plexbio Co., Ltd. Image differentiated multiplex assays
US10839509B2 (en) 2015-07-10 2020-11-17 3Scan Inc. Spatial multiplexing of histological stains
US11913024B2 (en) 2015-10-22 2024-02-27 Juno Therapeutics Gmbh Methods for culturing cells and kits and apparatus for same
US11492664B2 (en) 2016-06-17 2022-11-08 California Institute Of Technology Nucleic acid reactions and related methods and compositions
US10066263B2 (en) 2016-06-17 2018-09-04 California Institute Of Technology Nucleic acid reactions and related methods and compositions
US11796535B2 (en) 2016-09-16 2023-10-24 Plexbio Co., Ltd. Methods and systems for multiplex assays
US11866465B2 (en) 2017-04-27 2024-01-09 Juno Therapeutics Gmbh Oligomeric particle reagents and methods of use thereof
US11959856B2 (en) 2017-09-11 2024-04-16 California Institute Of Technology Multiplexing and quantification in PCR with reduced hardware and requirements

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