Assays%20of%20Immune%20Function - PowerPoint PPT Presentation

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Assays%20of%20Immune%20Function

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MHC-Ig dimer staining. For Function: ELISPOT. ICS. CD107 staining ... Advantages of Dimers vs. Tetramers. Dimers: Investigator can load peptide of interest ... – PowerPoint PPT presentation

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Title: Assays%20of%20Immune%20Function


1
Assays of Immune Function
2
Some Definitions
  • BrdU bromodeoxyuridine (incorporated into DNA
    during cell division)
  • CBA cytometric bead array
  • DC dendritic cell(s)
  • ELISA enzyme-linked immunosorbent assay
  • ICS intracellular cytokine staining
  • LPA lymphoproliferative assay (using
    3H-thymidine incorporation)
  • MHC major histocompatibility complex

3
Humoral versus Cellular Immunity
  • Humoral Immunity
  • Antibody production ELISA, CBA
  • Cellular Immunity
  • T cell specificity MHC multimer staining
  • Cytokines ELISA, CBA, ICS, ELISPOT
  • Degranulation CD107 staining
  • Cytotoxicity 51Cr release
  • Proliferation BrdU incorporation, LPA

4
Early Late Functions of Cellular Immunity
5
Categories of Cellular Assays
  • Bulk Assays
  • Radioactive
  • 51Cr release
  • LPA (3H-thymidine incorporation)
  • Non-Radioactive
  • ELISA
  • CBA
  • Single-cell Assays
  • For Specificity
  • MHC-peptide tetramer staining
  • MHC-Ig dimer staining
  • For Function
  • ELISPOT
  • ICS
  • CD107 staining
  • BrdU incorporation
  • CFSE assay

6
ELISA Assays
  • Require a matched pair of capture and detector
    antibodies for the analyte of interest
  • Wide variety of antibody pairs available for many
    different analytes
  • Standards available for assay calibration

7
CBA/Luminex Assays
  • Use multiplexed beads (varying in FL3/FL4
    intensity) labeled with capture antibodies for
    specific analytes
  • Sample (e.g., serum or cell culture supernatant)
    is added together with PE-labeled detector
    antibody
  • Software calculates the level of each analyte
    based on PE fluorescence of each bead population
    relative to a standard curve

8
CBA Assay Principle
9
Examples of CBA Assays
Spiking of single cytokines to show assay
specificity
Bead Intensity
Detector Ab Intensity
Day 4
Baseline
Effect of Rhinovirus inoculation on cytokines in
nasal lavage
IL-1b
IL-8
GM-CSF
IL-6
10
ELISA versus CBA Assays
ELISA CBA
Types of analytes antibodies, cytokines Antibodies, cytokines
Number of simultaneous analytes One Up to seven or more
Type of readout Colorimetric Flow cytometry
11
Best use of ELISA or CBA
  • ELISA defined system where only one or a few
    analytes are to be measured
  • Example testing the effect of various
    conditions on IL-12 production from purified DC
  • CBA systems in which multiple analytes are of
    potential interest and the sample is limited
  • Example measuring the effect of allergens on
    cytokines in human tears

12
MHC Multimer Assays
  • Measure binding of T cells to a specific
    peptideMHC combination
  • Can be used to identify rare populations of
    antigen-specific T cells without in vitro
    activation

13
MHC-peptide Dimers and Tetramers
14
Example of Dimer vs. Tetramer Staining
unloaded dimer
loaded dimer
tetramer
CMV tetramer
HLA-A2Ig
CD8 FITC
CD8 FITC
15
Advantages of Dimers vs. Tetramers
  • Dimers
  • Investigator can load peptide of interest
  • Can be used to coat plates for antigen-specific
    cell capture/stimulation
  • Tetramers
  • More MHC alleles commercially available
  • Higher affinity binding in some systems
  • Directly fluorochrome labeled

16
ELISPOT Assays
  • PBMC are plated on a filter-bottom 96-well plate
    coated with anti-cytokine antibody.
  • The plate is cultured 24-48 hours to allow
    cytokine secretion and capture on the plate.
  • Cells are washed off and detector antibody is
    added, followed by enzyme substrate.
  • Cytokine-secreting cells are identified as spots
    of secreted cytokine.

17
ELISPOT Assay Principle
Add PBMC
Wash out cells, add detector Ab
Prepare PBMC and count
wash
24 h
1 h
Coat plate with anti-cytokine Ab
Add Ag
15 min
Count on dissecting microscope or Analyze
on automated reader
Wash, add substrate
18
ICS Assays
  • Measure production of cytokines in short-term
    stimulated whole blood, PBMC, etc.
  • Can measure multiple cell-surface and
    intracellular markers in combination, using
    multiparameter flow cytometry
  • Can detect rare events such as antigen-specific T
    cells

19
Principle of Plate-Based ICS Assays
Incubate 6-24 h
20 ml PBMC/ WB sample
Antigenic stimulus brefeldin A
  • Fix cells
  • Permeabilize
  • Stain

Gate on cells of interest
20
Example of ICS Results
pp65 protein
peptide mix
A2 peptide
CMV lysate
CD4
CD69 PE
CD8
anti-IFNg FITC
21
Correlation of ICS and ELISPOT Assays
CMV pp65 peptide mix
CMV Lysate
1200
2000
2
r
0.4
2
r
0.3
900
1500
p lt 0.1
p lt 0.05
ICS
ICS
600
1000
300
500
0
0
0
100
200
300
400
0
100
200
300
400
500
ELISPOT
ELISPOT
22
Comparison of Ag-Specific T Cell Methods
23
CD107 Assays
  • CD107a and CD107b are proteins found in cytotoxic
    granules of CTL and other cells
  • Upon degranulation, CD107a and CD107b are
    transiently transported to the cell surface
  • Using labeled antibodies to CD107a and CD107b
    during short-term stimulation, the exocytosis of
    CD107 is captured on degranulating cells.

24
Example of CD107 Assay
25
BrdU Assays
  • Can measure cell proliferation based on
    incorporation of fluorescently labeled BrdU
  • Can be combined with cell-surface and
    intracellular markers (e.g., cytokines) for
    multiparameter staining

26
Principle of BrdU Assay (with IFNg)
27
Example of Ag-Specific BrdU Assay
p55 gag
HIV-REMUNE
Unstimulated
CD71PE
Anti-BrdU FITC/DNase
28
CFSE Assays
  • Cells (usually PBMC) are labeled with CFSE dye,
    then allowed to proliferate in vitro (or in vivo
    in mice)
  • CFSE is divided equally among daughter cells, so
    each generation becomes half as intense in CFSE
    staining

29
Example of CFSE Assay
Day 0
Day 4 - IL-4 cells
No CD81
CD81
30
Comparison of CFSE and BrdU Assays
unstimulated
SEB
CFSE
31
Categories of Cellular Assays
  • Bulk Assays
  • Radioactive
  • 51Cr release
  • LPA (3H-thymidine incorporation)
  • Non-Radioactive
  • ELISA
  • CBA
  • Single-cell Assays
  • For Specificity
  • MHC-peptide tetramer staining
  • MHC-Ig dimer staining
  • For Function
  • ELISPOT
  • ICS
  • CD107 staining
  • BrdU incorporation
  • CFSE assay
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