Mapping of the binding specificity for five monoclonal antibodies recognizing 3-deoxy-D-manno-octulosonic acid in bacterial lipopolysaccharides.

Mouse mAb were produced against the deep rough strains Salmonella minnesota R 595, Salmonella typhimurium SL 1102, and Escherichia coli D21f2 and screened by enzyme immunoassay against LPS of several chemotypes. Five antibodies were selected for their ability to bind to chemotype deep rough (Re) LPS which has two 3-deoxy-D-manno-octulosonic acid (Kdo) residues in its nonreducing end. Structurally verified oligosaccharides isolated from rough LPS and synthetic analogues of Kdo were used in an enzyme immunoassay inhibition test to determine the binding epitopes for the antibodies. According to their specificities, the antibodies could be divided into three groups. For two of the groups, the recognized structure was the alpha-Kdo (2----4) Kdo disaccharide and for one group the alpha-Kdo (2----4) alpha-Kdo beta-D-GlcN (1----6) alpha-D-GlcN tetrasaccharide, representing a partial structure of the Re LPS. Inhibition studies with synthetic analogues of Kdo showed that the anomeric configuration and the free carboxyl group of the Kdo residue are important features for antibody binding. Changes in the C-1 to C-6 region of the Kdo molecule do influence the antibody recognition considerably whereas changes in the exocyclic C-7 to C-8 region are of secondary importance. Calculation of the conformation of the inner core region showed that the alpha-Kdo (2----4) alpha-Kdo (2---- disaccharide was free and accessible in chemotype Re LPS, but that linkage of a L-glycero-D-manno-heptose to O-5 of the subterminal Kdo both changes the conformation of the Kdo-disaccharide and covers it thereby making it less accessible.