Real-time measurement of in vitro peptide binding to soluble HLA-A*0201 by fluorescence polarization.
Measuring the interaction of class I human leukocyte antigens (HLA) and their peptide epitopes acts as a guide for the development of vaccines, diagnostics, and immune-based therapies. Here, we report the development of a sensitive biochemical assay that relies upon fluorescence polarization to indicate peptide interactions with recombinant soluble HLA proteins. It is a cell- and radioisotope-free assay that has the advantage of allowing the direct, real-time measurement of the ratio between free and bound peptide ligand in solution without separation steps. Peptide/HLA assay parameters were established using several HLA A*0201-specific fluorescein isothiocyanate-labeled peptides. Optimal loading of synthetic peptides into fully assembled soluble HLA-A*0201 complexes was enabled by thermal destabilization at 53 degrees C for 15 min, demonstrating that efficient peptide exchange does not require the removal of endogenous peptides from the reaction environment. An optimal ratio of three beta-2 microglobulin molecules per single HLA heavy chain was determined to maximize peptide binding. Kinetic binding studies indicate that soluble HLA-A*0201/peptide interactions are characterized by a range of moderate k(on) values (1 x 10(4) to 8.7 x 10(4) M(-1) s(-1)) and slow k(off) values (1.9 x 10(-4) to 4.3 x 10(-4) s(-1)), consistent with parameters for native HLA molecules. Testing of the A*0201-specific peptides with 48 additional class I molecules demonstrates that the unique peptide binding behavior of individual HLA molecules is maintained in the assay. This assay therefore represents a versatile tool for characterizing the binding of peptide epitopes during the development of class I HLA-based vaccines and immune therapies.
Buchli
Rico
R
Pure Protein L.L.C., Oklahoma City, Oklahoma 73104-3698, USA. rico-buchli@ouhsc.edu
VanGundy
Rodney S
RS
Hickman-Miller
Heather D
HD
Giberson
Christopher F
CF
Bardet
Wilfried
W
Hildebrand
William H
WH
D003429:Cross Reactions; D004797:Enzyme-Linked Immunosorbent Assay; D000939:Epitopes / Q000378:metabolism; D016650:Fluorescein-5-isothiocyanate / Q000378:metabolism; D005454:Fluorescence Polarization / Q000379:methods / Q000592:standards; D015234:HLA-A Antigens / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D015789:HLA-A2 Antigen; D015235:HLA-B Antigens / Q000276:immunology / Q000378:metabolism; D015236:HLA-C Antigens / Q000276:immunology / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism*; D011485:Protein Binding; D011994:Recombinant Proteins / Q000138:chemical synthesis / Q000378:metabolism; D012995:Solubility; D001613:beta 2-Microglobulin / Q000737:chemistry / Q000378:metabolism
D000939:Epitopes; D015234:HLA-A Antigens; C435939:HLA-A*02:01 antigen; D015789:HLA-A2 Antigen; D015235:HLA-B Antigens; D015236:HLA-C Antigens; D010446:Peptide Fragments; D011994:Recombinant Proteins; D001613:beta 2-Microglobulin; D016650:Fluorescein-5-isothiocyanate