Determination of the epitope specificity of monoclonal antibodies against the inner core region of bacterial lipopolysaccharides by use of 3-deoxy- d- manno-octulosonate-containing synthetic antigens

Abstract

Partial structures of enterobacterial lipopolysaccharides (LPS) of the Rechemotype, consisting of lipid A and 3-deoxy- d- manno-2-octulosonic acid (Kdo), as well as oligosaccharides and derivatives of Kdo were synthesized and used to characterize the epitope specificity of monoclonal antibodies against Re-mutant LPS. High-molecular-weight antigens, obtained after copolymerization of the respective allyl glycosides with acrylamide, and the haptenic oligosaccharides were used in immunoprecipitation, immune hemolysis, and in inhibition assays. A monoclonal antibody (clone 20, IgM) recognizing a terminal Kdo p group was shown to require for its binding the α-anomeric configuration and OH-4 and OH-5 groups, whereas the C-7C-8 chain was of minor importance. Another monoclonal antibody (clone 25, IgG 3), which recognizes a (2→4)-linked Kdo disaccharide, was shown to require for its binding the α-anomeric configuration of both residues. The isomer having a reducing β-Kdo residue was significantly less active, and that with a terminal β-Kdo group was completely inactive. The OH-5 group of the reducing residue was shown to be not important for the specificity of this antibody, since it could be replaced by a hydrogen atom without loss of serological reactivity. The α-(2→8)-linked Kdo disaccharide was strongly cross-reactive with its (2→4)-linked isomer. The antibody recognized also parts of the 2-amino-2-deoxy- d-glucose backbone of lipid A.