Flow-cytometric determination of peptide-class I complex formation. Identification of p53 peptides that bind to HLA-A2.
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.
Zeh
H J
HJ
Department of Surgery, University of Pittsburgh, Pennsylvania.
Leder
G H
GH
Lotze
M T
MT
Salter
R D
RD
Tector
M
M
Stuber
G
G
Modrow
S
S
Storkus
W J
WJ
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*
D015789:HLA-A2 Antigen; D016159:Tumor Suppressor Protein p53