1. Real-Time Measurment of in Vitro Peptide Binding to Soluble HLA-A*0201 by Fluorescene Polarization.
- Author
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Buchli, Rico, VanGundy, Rodney S., Hickman-Miller, Heather D., Giberson, Christopher F., Bardet, Wilfried, and Hildebrand, William H.
- Subjects
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LEUCOCYTES , *PROTEINS , *PEPTIDES , *BIOMOLECULES , *VACCINATION , *HLA histocompatibility antigens - Abstract
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 °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 β-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 kon values (1 × 104 to 8.7 × 104 M-1 s-1) and slow koff values (1.9 × 10-4 to 4.3 × 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. [ABSTRACT FROM AUTHOR]
- Published
- 2004
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