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 identi fy a series of peptides derived from the human p53 tumor-suppressor ge ne product capable of binding the HLA-A2 class I allele. Brief pH 3.3 acid treatment of human cell lines rapidly denatures preexisting class I complexes, as detected by loss of binding of conformation-dependent mAbs, leaving only free class I heavy chains associated with the viab le cell surface. These heavy chains may be induced to refold and be re cognized by antibodies (in 2-4 hours) when acid-treated cells are coin cubated with exogenous beta(2)-microglobulin and peptides capable of b inding the relevant class I allele examined. This assay, with a detect ion limit of 1-10 nM peptide, was used to screen the capacity of a pan el of nine peptides bearing HLA-A2-binding motifs and derived from the human p53 tumor-suppressor protein sequence. Eight of the nine peptid es bound to, and reconstituted, HLA-A2 on acid-treated cells. This ass ay 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.