title: Stability screening of arrays of major histocompatibility complexes on combinatorially encoded flow cytometry beads.
abstract: The binding and stabilization capacity of potential T cell epitopes to class I MHC molecules form the basis for their immunogenicity and provide fundamental insight into factors that dictate cellular immune responses. We have developed a versatile high throughput cell-free method to measure MHC stability by capturing a variety of MHC products on the surface of streptavidin-coated particles followed by flow cytometry analysis. Arrays of peptide-MHC combinations, generated by exchanging conditional ligand-loaded MHC, could be probed in a single experiment, thus combining the molecular precision of biochemically purified MHCs with high content multiparametric flow cytometry-based assays. Semiquantitative determination of the peptide affinity for the restriction element could also be accomplished through competition experiments using this bead-based assay. Furthermore, the generated peptide-MHC reagents could directly be applied to antigen-specific CD8(+) T lymphocyte analysis. The combinatorial labeling of beads allowed straightforward identification by their unique fluorescent signatures and provided a convenient means for extended assay multiplexing.
authors.affiliation: Department of Microbiology, Department of Biological Sciences, and Immunology Programme, Life Sciences Institute, National University of Singapore, 117456 Singapore.
authors.forename: Shi Ling
authors.initials: SL
authors.lastname: Chew
authors.forename: Ming Yan
authors.initials: MY
authors.lastname: Or
authors.forename: Cynthia Xin Lei
authors.initials: CX
authors.lastname: Chang
authors.forename: Adam J
authors.initials: AJ
authors.lastname: Gehring
authors.forename: Antonio
authors.initials: A
authors.lastname: Bertoletti
authors.forename: Gijsbert M
authors.initials: GM
authors.lastname: Grotenbreg
mesh_terms: D018414:CD8-Positive T-Lymphocytes / Q000737:chemistry* / Q000166:cytology / Q000276:immunology; D002460:Cell Line; D002474:Cell-Free System / Q000737:chemistry / Q000276:immunology; D018984:Epitopes, T-Lymphocyte / Q000737:chemistry* / Q000276:immunology; D005434:Flow Cytometry / Q000379:methods*; D015395:Histocompatibility Antigens Class I / Q000737:chemistry* / Q000276:immunology; D006801:Humans; D019151:Peptide Library
chemical_list: D018984:Epitopes, T-Lymphocyte; D015395:Histocompatibility Antigens Class I; D019151:Peptide Library