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Author Notes:

Correspondence: John D. Altman, Emory Vaccine Center, 954 Gatewood Road, Atlanta, GA 30329; Phone: (404) 727-5981; Email: jaltman@rmy.emory.edu

Acknowledgments: We thank Takeshi Sano (Harvard Medical School) for the StvC expression plasmid and advice on StvC refolding and purification.

This paper is dedicated to the memory of Vitalii Grigoriev.

Subject:

Research Funding:

This work was supported by the NIAID MHC Tetramer Core Contract (N01-AI-25456).

Keywords:

  • MHC tetramers
  • streptavidin
  • flow cytometry

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

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Journal Title:

Journal of Immunological Methods

Volume:

Volume 319, Number 1-2

Publisher:

, Pages 13-20

Type of Work:

Article | Post-print: After Peer Review

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.

Copyright information:

© 2006 Elsevier B.V. All rights reserved.

This is an Open Access work distributed under the terms of the Creative Commons Attribution-NonCommerical-NoDerivs 3.0 Unported License (http://creativecommons.org/licenses/by-nc-nd/3.0/).

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