An HLA class I peptide-binding assay based on competition for binding to class I molecules on intact human B cells: Identification of conserved HIV-1 polymerase peptides binding to HLA-A ∗0301

Abstract

A peptide-binding assay employing the HLA class I molecules on intact human B cells is described. The peptide antigens are stripped from the HLA class I molecules by mild acid treatment, after which the cells are incubated with a FL-labeled reference peptide together with different concentrations of the peptide of interest. The effectiveness by which the latter peptide competes for binding to the HLA class I molecules is assayed by measuring the amount of HLA-bound FL-labeled reference peptide with FACscan analysis. The assay is easy to perform because there is no need to purify HLA class I molecules, or to transfect cells with HLA class I molecules, and no radioactive label is used. Moreover, large panels of HLA-typed human B-cell lines are available as tools for peptide binding to a vast array of HLA molecules. The binding assay was optimized and validated with peptides of known binding capacity to either HLAA ∗0201 or HLA-A ∗0301. The kinetics of peptide binding in this assay were shown to be comparable to that in assays employing soluble HLA class I molecules. Application of the assay in the search for potential HLAA ∗0301 restricted CTL epitopes, derived from HIV-1 polymerase, resulted in the identification of five highaffinity binding peptides.