This proposal continues a long term study of the structure and specificity of antibodies to a homopolymer of glucose alpha(1-6) dextran and its oligosaccharides, and on blood group A, B and H substances. It characterizes their primary and higher order structures by a variety of techniques. The cavity-and groove-type sites were defined, the former specific for terminal non-reducing alpha(1-6) linked glucoses plus 3 or 4 additional residues, and the latter reacting with internal chains of up to six or seven glucoses not including the non-reducing end. Anti-idiotypic sera and monoclonal antibodies recognizing either groove-or cavity-type sites have been obtained. Model-building studies show that two cavity-type sites vary in size. The hybridoma repertoire of anti-alpha(1-6) dextrans is being compared with E. coli expressed Fab repertoire. The role of Vl-CDR3 and J(kappa) on antigen binding, idiotype expression and combining sites structure of the groove-type anti-alpha(1-6) dextrans with V(kappa)-Oxl light chain is being studied by the generation of recombinant Fab and computer modelling. %%% The research will concentrate on the following aspects of monoclonal antibodies to .(1-->6) dextrans: a) Mapping the structures of the combining sites by immunochemical methods and studies of anti-.(1-->6) dextrans with cavity and groove type antibody combining sites and their idiotypic specificities; b) Providing quantities of the various characterized anti-.(1-->6) dextrans to various crystallographic laboratories for crystallization and high resolution x-ray diffraction studies. c) Continuation of replacement studies of a cloned antibody (amino acid residues 31 to 35) by glycines that have shown an extraordinary effect in greatly reducing anti .(1-->6) dextran reactivity and substantial restoration of 80% of the original anti .(1-->6) dextran specificity by replacing glycine 32 and 33 by tyrosine and tryptophan. d) Studying the effects of similar substitutions in other interaction domains of the antibody.
