A robust method for production of MHC tetramers with small molecule fluorophores.

Tetramers of major histocompatibility complex molecules (MHC) are now well-established reagents for the detection of antigen-specific T cells by flow cytometry. MHC tetramers are prepared by mixing enzymatically biotinylated MHC molecules with commercial preparations of streptavidin, usually conjugated to a fluorescent phycobiliprotein such as phycoerythrin (PE) or allophycocyanin (APC). While data obtained with MHC tetramers prepared with small molecule fluorophores has been reported, considerable lot-to-lot variation among conventional streptavidin conjugates to small molecules prevents routine preparation of such reagents. We now report robust preparation of MHC tetramers with small molecule fluorophores, using a recombinant mutant of streptavidin incorporating a carboxy-terminal cysteine in each of the four identical subunits that is conjugated to maleimide derivatives of any of several small molecule fluorophores. These reagents significantly expand the versatility of the MHC tetramer methodology.