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Antigen Binding: Tetramer MHC-Peptide

What this assay tells you

Both B and T lymphocytes have membrane receptors specific for antigen: BCR and TCR. Antigen binding to these receptors activates the lymphocytes to become effector and memory cells.

BCR binds native antigen directly. Binding can be detected using fluorescent antigen and flow cytometry, radioactive antigen and autoradiography, or enzyme-linked antigen and histochemistry.

TCR binds peptide on syngeneic (self) MHC: CD8 T cells use TCR to bind peptide on Class I MHC, CD4 T cells use TCR to bind peptide on on Class II MHC. Single MHC-peptide complexes bind TCR + co-receptor with too low an avidity to be detected. Tetramers of MHC-peptide bind with a high enough avidity to allow specific peptide-binding T cells to be detected. Flow cytometry is generally used to count tetramer MHC-peptide-binding T cells.

What you need to do the assay

• Cell suspension containing T lymphocytes.
• Tetramer MHC of the appropriate class (Class I for CD8 T cells and Class II for CD4 T cells), allotype (syngeneic MHC), and peptide-binding specificity loaded with the specific peptide and tagged with a fluorochrome.
• Flow cytometer.

How the assay is done

• Incubate the cells with the labeled tetramer MHC-peptide; wash.
• Analyze in flow cytometer.
• Negative controls use another peptide or MHC allele. Positive controls use T cell lines of known specificity for the tetramer MHC-peptide.

How to interpret the results

Higher mean fluorescence intensity indicates more specific MHC-peptide binding (see Flow Cytometry).

Additional comments

Frequencies of specific antigen-binding cells are too low to be detectable in naïve lymphocyte populations. This assay is generally only done with populations of memory T cells where the frequency of specific cells has been increased by immunization or infection.

Useful web sites:

http://www.probes.com/handbook/boxes/1642.html