title: Flow-cytometric determination of peptide-class I complex formation. Identification of p53 peptides that bind to HLA-A2.
abstract: A novel class I-peptide-binding assay was developed and used to identify a series of peptides derived from the human p53 tumor-suppressor gene product capable of binding the HLA-A2 class I allele. Brief pH 3.3 acid treatment of human cell lines rapidly denatures pre-existing class I complexes, as detected by loss of binding of conformation-dependent mAbs, leaving only free class I heavy chains associated with the viable cell surface. These heavy chains may be induced to refold and be recognized by antibodies (in 2-4 hours) when acid-treated cells are coincubated with exogenous beta 2-microglobulin and peptides capable of binding the relevant class I allele examined. This assay, with a detection limit of 1-10 nM peptide, was used to screen the capacity of a panel of nine peptides bearing HLA-A2-binding motifs and derived from the human p53 tumor-suppressor protein sequence. Eight of the nine peptides bound to, and reconstituted, HLA-A2 on acid-treated cells. This assay system will enable the rapid identification of peptides binding to any class I allele, which is the initial prerequisite for elucidating potential CD8+ T-cell epitopes.
authors.affiliation: Department of Surgery, University of Pittsburgh, Pennsylvania.
authors.forename: H J
authors.initials: HJ
authors.lastname: Zeh
authors.forename: G H
authors.initials: GH
authors.lastname: Leder
authors.forename: M T
authors.initials: MT
authors.lastname: Lotze
authors.forename: R D
authors.initials: RD
authors.lastname: Salter
authors.forename: M
authors.initials: M
authors.lastname: Tector
authors.forename: G
authors.initials: G
authors.lastname: Stuber
authors.forename: S
authors.initials: S
authors.lastname: Modrow
authors.forename: W J
authors.initials: WJ
authors.lastname: Storkus
mesh_terms: D000595:Amino Acid Sequence; D002460:Cell Line; D005434:Flow Cytometry; D015789:HLA-A2 Antigen / Q000032:analysis*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D008969:Molecular Sequence Data; D011485:Protein Binding; D011487:Protein Conformation; D013601:T-Lymphocytes / Q000276:immunology*; D016159:Tumor Suppressor Protein p53 / Q000032:analysis / Q000276:immunology*
chemical_list: D015789:HLA-A2 Antigen; D016159:Tumor Suppressor Protein p53