Tetramer Assays
A MHC tetramer assay or simply tetramer assay is a procedure of use labeled tetrameric protein to detect and quantify T-cells that are specific for a given antigen within blood or tissue samples. The IML is able to produce both MHC class I and class II tetramers for use in both mouse and human systems, which can be adapted for detection by either flow cytometry or CyTOF.
MHC class II monomers are produced in a mammalian expression system by linking the peptide of interest to the gene of N-terminal of β-chain and co-transfecting with α-chain. All MHC class I monomers produced by the IML are folded using a photocleavable ligand. Upon exposure to UV light, the photocleavable ligand is broken thereby allowing your peptide of interest to be exchanged into the binding site of the MHC class I monomer. The peptide-exchanged class I monomer and class II monomer are then converted to tetramers by labeling with streptavidin conjugated fluorochrome and used for cell staining. The use of combinatorial coding of tetramers allows for high throughput parallel detection of antigen-specific T cells and also greatly decreases non-specific background within samples thereby allowing visualization of small tetramer-positive populations within a single sample.
Useful Links
- Available MHC Class I Products
- Protocol for tetramer generation and cell staining: Multimer_Staining_IML_SOP
- Solubility Guideline for peptide: A Guide to Handling and Storing-Peptides
- Reference for bar-coding of HLA staining: https://www.nature.com/articles/nmeth.1345.pdf
- Sources of fluorochromes
BioLegend: https://www.biolegend.com/immunobiology
BD Horizon: http://www.bdbiosciences.com/us/s/brilliantdyes
Invitrogen: https://www.thermofisher.com/us/en/home/life-science/cell-analysis/flow-cytometry.html