The long term goal of this project is to determine the relationship between diversity and complementarity and to determine the repertoire and fine specificity of combining sites to a single well defined antigenic determinant, the site filling epitope of alpha(1 ->6)dextran. We have studied a series of hybridomas specific for alpha(1->6)dextran, characterized their antibodies immunochemically as to fine structure and idiotypic specificity, and determined the primary structures of their VH and VL. Modeling of cavity- and groove-type sites, will be continued to determine variations in the sites in relation to sequence differences. An ultimate objective is to determine the three dimensional xray structure of their combining sites. Studies with hybridomas have shown that they do not comprise the totality of antibody specificities seen in serum. Thus there is a hidden portion of the repertoire which can only be discovered by the use of multiple strains, varied conditions of immunization etc. This is important in determining the extent of germ line usage in evaluating the repertoires. Different VH and VL can generate similar binding specificities and the presence of N-linked carbohydrate in VH influences the strength of the antigen-antibody interaction; thus not only the amino acid sequence of the variable region but also the carbohydrate present there is important for determining binding. Site directed mutagenesis will be coupled with gene transfection techniques to evaluate the contributions of residues in VH and VL to specificity and binding. We have studied a monoclonal anti-blood group A in humans and mice and find that identity in fine structure can occur with different residues in the CDRs of the two species; this will be expanded to define the repertoire in two different species to anti-B as well anti-A in relation to identities and differences in CDRs and fine specificity of the sites. We shall compare the hybridoma repertoire in great detail with that produced in E. coli by the Lerner procedure with respect to cavity- vs groove-type sites, binding constants, site sizes, fine structure using various oligo- and fluorodeoxy oligosaccharides, and idiotype specificities. The findings will establish whether the two repertoires are drawn from the same universe of antibody combining sites.
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