Reagents and instruments for immunology, cell biology and molecular biology.

Human tetramers - Class I - FITC

Preparation of MHC Class I Tetramer Reagents

MHC class I tetramer reagents are generated primarily using the method developed by Altman et al., who demonstrated that tetramers of MHC class I/peptide complexes could be used as probes for detection and quantitation of antigen-specific CTL (Science 1996, 274: 94-96)

E. coli-expressed recombinant MHC class I heavy chain and β2-microglobulin (β2m) are folded in the presence of a peptide antigen to generate a soluble monomeric MHC class I/peptide complex (monomer). The monomer is then biotinylated by the biotin ligase enzyme, BirA, at a lysine residue present in the biotinylation sequence that has been added to the C-terminus of MHC class I heavy chain. The biotinylated monomer is purified by column chromatography. Purified biotinylated monomers are tetramerized by mixing with a fluorescently-labeled streptavidin to make MHC tetramer reagents.

MHC tetramers are labeled with fluorescent molecules including phycoerythrin (PE), allophycocyanin (APC), or Brilliant Violet 421 (BV421) and thus allow detection of antigen-specific T cells by flow cytometry or fluorescence microscopy.