A novel, highly efficient peptide-HLA class I binding assay using unfolded heavy chain molecules: identification of HIV-1 derived peptides that bind to HLA-A*0201 and HLA-A*0301.
A novel cell-free, highly automated peptide-HLA binding assay has been designed during which a mixture of unfolded recombinant HLA heavy chain molecules, beta 2-microglobulin and a fluorescent labeled standard peptide is allowed to form peptide-HLA complexes. The binding of a peptide of interest is monitored as the ability to inhibit the formation of fluorescent peptide-HLA complexes. The assay was validated using published, known HLA-A* 0201 and HLA-A* 0301 binding peptides. In addition a selected set of HIV-1LAI reverse transcriptase derived 10-mer peptides, that had been selected on the basis of HLA-A* 0201 or HLA-A* 0301 binding motifs, were tested for HLA-A* 0201/A* 0301 binding. In that set we identified 8 peptides which bound with high affinity to HLA-A* 0201 and 5 peptides which bound with high affinity to HLA-A* 0301. The major advantage of the use of denatured heavy chain is the improved economy and efficiency, as unfolded protein material is in principle easily accessible by recombinant technology.
Tan
T L
TL
Department of Immunohematology and Blood Bank, Leiden University Hospital, Netherlands.
Geluk
A
A
Toebes
M
M
Ottenhoff
T H
TH
Drijfhout
J W
JW
D054303:HIV Reverse Transcriptase / Q000235:genetics / Q000378:metabolism*; D015234:HLA-A Antigens / Q000737:chemistry / Q000378:metabolism*; D006801:Humans; D007118:Immunoassay / Q000379:methods*; D009842:Oligopeptides / Q000235:genetics / Q000378:metabolism; D010446:Peptide Fragments / Q000235:genetics / Q000378:metabolism; D011485:Protein Binding; D011489:Protein Denaturation; D001613:beta 2-Microglobulin / Q000378:metabolism
D015234:HLA-A Antigens; D009842:Oligopeptides; D010446:Peptide Fragments; D001613:beta 2-Microglobulin; D054303:HIV Reverse Transcriptase