title: One-pot, mix-and-read peptide-MHC tetramers.
abstract: BACKGROUND
  Cytotoxic T Lymphocytes (CTL) recognize complexes of peptide ligands and Major Histocompatibility Complex (MHC) class I molecules presented at the surface of Antigen Presenting Cells (APC). Detection and isolation of CTL's are of importance for research on CTL immunity, and development of vaccines and adoptive immune therapy. Peptide-MHC tetramers have become important reagents for detection and enumeration of specific CTL's. Conventional peptide-MHC-tetramer production involves recombinant MHC production, in vitro refolding, biotinylation and tetramerization; each step followed by various biochemical steps such as chromatographic purification, concentration etc. Such cumbersome production protocols have limited dissemination and restricted availability of peptide-MHC tetramers effectively precluding large-scale screening strategies involving many different peptide-MHC tetramers.
  
  
  RESULTS
  We have developed an approach whereby any given tetramer specificity can be produced within 2 days with very limited effort and hands-on time. The strategy is based on the isolation of correctly oxidized, in vivo biotinylated recombinant MHC I heavy chain (HC). Such biotinylated MHC I HC molecules can be refolded in vitro, tetramerized with streptavidin, and used for specific T cell staining-all in a one-pot reaction without any intervening purification steps.
  
  
  CONCLUSIONS
  We have developed an efficient "one-pot, mix-and-read" strategy for peptide-MHC tetramer generation, and demonstrated specific T cell straining comparable to a commercially available MHC-tetramer. Here, seven peptide-MHC tetramers representing four different human MHC (HLA) class I proteins have been generated. The technique should be readily extendable to any binding peptide and pre-biotinylated MHC (at this time we have over 40 different pre-biotinylated HLA proteins). It is simple, robust, and versatile technique with a very broad application potential as it can be adapted both to small- and large-scale production of one or many different peptide-MHC tetramers for T cell isolation, or epitope screening.
authors.affiliation: Laboratories of Experimental Immunology, University of Copenhagen, Copenhagen, Denmark.
authors.forename: Christian
authors.initials: C
authors.lastname: Leisner
authors.forename: Nina
authors.initials: N
authors.lastname: Loeth
authors.forename: Kasper
authors.initials: K
authors.lastname: Lamberth
authors.forename: Sune
authors.initials: S
authors.lastname: Justesen
authors.forename: Christina
authors.initials: C
authors.lastname: Sylvester-Hvid
authors.forename: Esben G
authors.initials: EG
authors.lastname: Schmidt
authors.forename: Mogens
authors.initials: M
authors.lastname: Claesson
authors.forename: Soren
authors.initials: S
authors.lastname: Buus
authors.forename: Anette
authors.initials: A
authors.lastname: Stryhn
mesh_terms: D019921:Biotinylation; D018604:Epitope Mapping; D015395:Histocompatibility Antigens Class I; D006801:Humans; D007158:Immunologic Techniques; D008285:Major Histocompatibility Complex; D008722:Methods; D010455:Peptides / Q000276:immunology / Q000302:isolation & purification*; D019809:Streptavidin; D013602:T-Lymphocytes, Cytotoxic / Q000276:immunology*
chemical_list: D015395:Histocompatibility Antigens Class I; D010455:Peptides; D019809:Streptavidin