AN HLA CLASS-I PEPTIDE-BINDING ASSAY BASED ON COMPETITION FOR BINDINGTO CLASS-I MOLECULES ON INTACT HUMAN B-CELLS - IDENTIFICATION OF CONSERVED HIV-1 POLYMERASE PEPTIDES BINDING TO HLA-A-ASTERISK-0301

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 a re incubated with a FL-labeled reference peptide together with differe nt concentrations of the peptide of interest. The effectiveness by whi ch the latter peptide competes for binding to the HLA class I molecule s 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 cell s with HLA class I molecules, and no radioactive label is used. Moreov er, large panels of HLA-typed human B-cell lines are available as tool s for peptide binding to a vast array of HLA molecules. The binding as say was optimized and validated with peptides of known binding capacit y to either HLA-A0201 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. Appli cation of the assay in the search for potential HLA-A0301 restricted CTL epitopes, derived from HIV-1 polymerase, resulted in the identification of five high affinity bindi ng peptides.