Tanigaki N, Fruci D, Chersi A, Butler R H
Institute of Cellular Biology, CNR, Rome, Italy.
Hum Immunol. 1993 Feb;36(2):119-27. doi: 10.1016/0198-8859(93)90114-g.
Unfolded HLA class I alpha chains were isolated from B-cell lysates by alkaline denaturation and subsequent gel filtration and used for the detection of HLA class-I-peptide binding. Binding to specific peptides in the presence of excess beta 2-microglobulin induced the unfolded alpha chains to refold and acquire a conformation that is specific to folded alpha chains. This conformational change was measured by a specific RIA that involves inhibition of the binding of 125I-labeled HLA-A2 alpha/beta dimers and rabbit anti-HLA-B7 serum absorbed with beta 2-microglobulin. This assay procedure does not require labeling of either test peptides or test class I proteins and does not seem to have specificity degeneracy. It is applicable to the detection of peptide binding by all HLA class I allelic proteins. Evaluation of the assay conditions and HLA allelic specificity of the peptide binding defined by the use of synthetic peptides are described here, including the technical details, specificity, and reproducibility.
