The physicochemical parameters which guide antibody recognition of a protein antigen will be studied employing cytochrome c (cyt) as a model antigen. Of particular interest is the basis for differences in the antibody repertoires responding to variant cyts. Monoclonal antibodies (mAbs) will be elicited against several cyts that differ by as little as one amino acid in an antigenic region. The epitopes recognized will be determined from antibody binding assays employing a number of cyt variants prepared by site-directed mutagenesis or obtained from natural sources. The amino acid sequences of the heavy and light chains of the mAbs will be deduced from the nucleotide sequences of cDNA. mAbs binding similar epitopes will be compared in terms of primary structure and any differences that occur as a result of an amino acid change in an epitope will be determined. The three-dimensional structures of mAbs showing such differences will be modeled using computer graphics as will their interaction with cyt to obtain insight into the chemistry of the interaction. Some of these models will be tested by X-ray crystallography in a related study. Preliminary data indicate relatively restricted antibody gene usage with distinct V region genes used in response to the two major antigenic regions of cyt. An effect of epitope topography on complementary determining region length is also apparent. These observations will be studied further to advance our understanding of antibody recognition of native protein antigens. The results should serve as a model for protein-protein interactions, in general.
