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.
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 HLA-A*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 HLA-A*0301 restricted CTL epitopes, derived from HIV-1 polymerase, resulted in the identification of five high-affinity binding peptides.
van der Burg
S H
SH
Department of Immunohaematology and Bloodbank, University Hospital, Leiden, The Netherlands.
Ras
E
E
Drijfhout
J W
JW
Benckhuijsen
W E
WE
Bremers
A J
AJ
Melief
C J
CJ
Kast
W M
WM
D000595:Amino Acid Sequence; D001402:B-Lymphocytes / Q000276:immunology* / Q000378:metabolism*; D001665:Binding Sites; D001667:Binding, Competitive; D017124:Conserved Sequence; D000939:Epitopes / Q000235:genetics / Q000378:metabolism; D015488:HIV Antigens / Q000235:genetics / Q000378:metabolism; D054303:HIV Reverse Transcriptase; D015497:HIV-1 / Q000201:enzymology / Q000235:genetics; D015234:HLA-A Antigens / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D008969:Molecular Sequence Data; D027383:Papillomaviridae / Q000235:genetics / Q000378:metabolism; D010446:Peptide Fragments / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D011485:Protein Binding; D012194:RNA-Directed DNA Polymerase / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D013602:T-Lymphocytes, Cytotoxic / Q000276:immunology; D014764:Viral Proteins / Q000235:genetics / Q000378:metabolism
D000939:Epitopes; D015488:HIV Antigens; D015234:HLA-A Antigens; D010446:Peptide Fragments; D014764:Viral Proteins; D054303:HIV Reverse Transcriptase; D012194:RNA-Directed DNA Polymerase