A QUANTITATIVE ASSAY TO MEASURE THE INTERACTION BETWEEN IMMUNOGENIC PEPTIDES AND PURIFIED CLASS-I MAJOR HISTOCOMPATIBILITY COMPLEX-MOLECULES

A direct and sensitive biochemical assay to measure the interaction in solution between peptides and affinity-purified major histocompatibility complex (MHC) class I molecules has been generated. Specific binding reflecting the known class I restriction of cytotoxic T cell responses was obtained. Adding an excess of beta(2)-microglobulin (beta(2)m) significantly increased the rate of peptide association, but it did not affect the rate of dissociation. Binding was complicated by a rapid and apparently irreversible loss of functional MHC class I at 37 degrees C which might limit the life span of empty MHC class I thereby preventing the inadvertent exchange of peptides at the target cell surface. All class I molecules tested bound peptides of the canonical octa- to nona-meric length. However, one class I molecule, K-k, also bound peptides, which were much longer suggesting that the preference of class I molecules for short epitopes is not absolute and may be caused by factors other than the peptide-MHC class I binding event itself.