A Robust Method for Production of MHC Tetramers with Small Molecule Fluorophores

• Type of Material: Article
• Authors: Vasanthi Ramachandiran, Emory University; Vitalii Grigoriev, Emory University; Lan Lan, Emory University; Eugene Ravkov, Emory University; Suzanne A. Mertens, Emory University; John D Altman, Emory University
• Language: English
• Date: 2007-01-30
• Publisher: Elsevier
• Publication Version: Author Accepted Manuscript (After Peer Review)
• Copyright Statement: © 2006 Elsevier B.V. All rights reserved.
• License: Creative Commons Attribution-NonCommerical-NoDerivs 3.0 Unported
• Final Published Version (URL): http://dx.doi.org/10.1016/j.jim.2006.08.014
• Title of Journal or Parent Work: Journal of Immunological Methods
• ISSN: 0022-1759
• Volume: 319
• Issue: 1-2
• Start Page: 13
• End Page: 20
• Grant/Funding Information: This work was supported by the NIAID MHC Tetramer Core Contract (N01-AI-25456).
• Keywords: streptavidin; MHC tetramers; flow cytometry
• Research Categories: Biology, Microbiology

Abstract

Tetramers of major histocompatability 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.