Assays%20of%20Immune%20Function

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Assays of Immune Function
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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

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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

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Early Late Functions of Cellular Immunity
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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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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

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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

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CBA Assay Principle
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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
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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
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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

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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

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MHC-peptide Dimers and Tetramers
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Example of Dimer vs. Tetramer Staining
unloaded dimer
loaded dimer
tetramer
CMV tetramer
HLA-A2Ig
CD8 FITC
CD8 FITC
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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

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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.

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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
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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

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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
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Example of ICS Results
pp65 protein
peptide mix
A2 peptide
CMV lysate
CD4
CD69 PE
CD8
anti-IFNg FITC
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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
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Comparison of Ag-Specific T Cell Methods
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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.

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Example of CD107 Assay
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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

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Principle of BrdU Assay (with IFNg)
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Example of Ag-Specific BrdU Assay
p55 gag
HIV-REMUNE
Unstimulated
CD71PE
Anti-BrdU FITC/DNase
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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

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Example of CFSE Assay
Day 0
Day 4 - IL-4 cells
No CD81
CD81
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Comparison of CFSE and BrdU Assays
unstimulated
SEB
CFSE
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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