Antibody combining sites and oligosaccharide determinants studied by competitive binding, sequencing and X-ray crystallography
Synthetic o ligosaccharides and polysaccharides of known structure have been covalently attached to carrier protein and enzymes for use in a strategy to generate and select monoclonal antibodies with well defined carbohydrate binding profiles. Direct c ompetitive binding assays utilizing antibody bound to a solid phase and saccharide-enzym e conjugates provided convenient and reliable measurement of specif icity. Protocols were developed to prepare these carbohydrate-enz yme conjugates. Antibodies specific for the Brucella A and M polysaccharide/a ntigen were selected and characterized by these techniques. The crystal s tructure of an Fab fragment derived from o ne antibody has been solved at 2.7A resolution. Preliminary crystal s tructure data in conjunction with the amino acid sequence of light and Fd polypeptide chains imp1 icate tyrosine, arg i n i ne, asparag ine, glutamine and aspartic acid as the residues that contact antigen. A second antibody-antigen system involving the Shigella flexneri Y antigen has been studied by a similar approach, although in this case two distinct antibodies have been subjected to detailed binding studies with oligosaccharide fragments of the polysaccharide repeating unit and specifically modified derivatives. These data support the contention that the principal polar interactions between an oligosaccharide and its antibody involve a small group of hydroxyl residues and that specifically deoxygenated oligosaccharide inhibitors can have association constants significantly higher t han the natural ol igosacc har ide .