Flow-cytometric determination of peptide-class I complex formation. Identification of p53 peptides that bind to HLA-A2

Hum Immunol. 1994 Feb;39(2):79-86. doi: 10.1016/0198-8859(94)90105-8.


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.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Amino Acid Sequence
  • Cell Line
  • Flow Cytometry
  • HLA-A2 Antigen / analysis*
  • Humans
  • Hydrogen-Ion Concentration
  • Molecular Sequence Data
  • Protein Binding
  • Protein Conformation
  • T-Lymphocytes / immunology*
  • Tumor Suppressor Protein p53 / analysis
  • Tumor Suppressor Protein p53 / immunology*


  • HLA-A2 Antigen
  • Tumor Suppressor Protein p53