PEPTIDE BINDING TO THE MOST FREQUENT HLA-A CLASS-I ALLELES MEASURED BY QUANTITATIVE MOLECULAR-BINDING ASSAYS

Quantitative assays to measure the binding of defined synthetic antige nic peptides and purified MHC class I molecules are described for seve ral common human HLA-A alleles (A1, A2.1, A3, A11 and A24). Under appr opriate conditions, the binding of radiolabeled peptides to purified M HC class I molecules is very effective, highly specific, and appears t o be dependent on the specific sequence motif of the peptide as define d by critical anchor residue positions. Establishment and optimization of the assay reveals that a relatively high fraction of the MHC class I molecules isolated from EBV transformed B cell line sources is capa ble of binding exogenously added peptide. Scatchard analysis for all a lleles yields 5-10% occupancy values. There is a stringent peptide siz e requirement that is reflected by the direct influence of peptide len gth on the binding affinity. The peptide-MHC class I interactions demo nstrate remarkable similarity to peptide-MHC class II interactions, bo th in overall affinity and kinetic behavior. The immunological relevan ce of the peptide-MHC class I binding assay is also demonstrated by me asuring the affinity of a panel of previously described HLA restricted peptides for their HLA restriction element. In 91% (10/11) of the cas es, the peptides bound with affinities of 50 nM or less, and in the re maining 9% (1/11) of the cases, in the 50 to 500 nM range. Thus, these data provide the first quantitative estimate of what level of HLA-A b inding affinity is associated with a diverse panel of immunodominant C TL epitopes in man.