Binding characteristics and cross-reactivity of three different antilipid A monoclonal antibodies.
A detailed characterization of binding specificity and cross-reactivity of three antilipid A murine mAb was performed. Binding characteristics of these three mAb were investigated against Ag (ReLPS, lipid A, derivatives of lipid A) in solid phase (ELISA) and in fluid phase (C consumption, inhibition studies), and upon incorporation in membranes (E: passive hemolysis assay, and liposomes: inhibition studies). Cross-reactivity with heterologous Ag was investigated in ELISA (LPS, Gram-negative bacteria) and immunoblot experiments (LPS). The binding specificity of mAb 26-5 (IgG2b), raised against synthetic lipid A, was located in the hydrophilic region of biphospholipid A and was also exposed after membrane incorporation of lipid A or after preincubation of lipid A with polymyxin B (PMX). mAb 26-20 (IgM), also raised against synthetic lipid A, showed binding specificity for the hydrophobic region of lipid A: no binding to membrane-associated lipid A could be demonstrated, and binding in ELISA could be blocked very efficiently by PMX. The reaction pattern of mAb 8-2 (IgM), raised against the heat-killed Re mutant of Salmonella typhimurium, was in part similar to that of mAb 26-20. However, inhibition of binding with PMX was less efficient and a high specificity for ReLPS, also after membrane incorporation of this Ag, was demonstrated. In contrast to mAb 26-5 and 26-20, mAb 8-2 showed extensive cross-reactivity with heterologous LPS preparations and heat-killed as well as live Gram-negative bacteria. It is concluded that each of the three mAb binds to a different antigenic epitope in lipid A and that exposure of those epitopes for antibody binding is restricted in a differential manner, depending on mode of Ag presentation. The here defined reaction patterns provide a basis for the interpretation of potential inhibitory effects on in vitro and in vivo biologic (and toxic) activities of endotoxins and Gram-negative bacteria.
Erich
T
T
Eijkman-Winkler Laboratory for Microbiology, University of Utrecht, The Netherlands.
Schellekens
J
J
Bouter
A
A
Van Kranen
J
J
Brouwer
E
E
Verhoef
J
J
D000818:Animals; D000911:Antibodies, Monoclonal / Q000096:biosynthesis / Q000378:metabolism*; D000918:Antibody Specificity; D001666:Binding Sites, Antibody; D001667:Binding, Competitive; D003165:Complement System Proteins / Q000378:metabolism; D003429:Cross Reactions; D004797:Enzyme-Linked Immunosorbent Assay; D004926:Escherichia coli / Q000276:immunology; D006090:Gram-Negative Bacteria / Q000276:immunology; D006461:Hemolysis; D006358:Hot Temperature; D015151:Immunoblotting; D008050:Lipid A / Q000276:immunology* / Q000378:metabolism; D008070:Lipopolysaccharides / Q000276:immunology; D051379:Mice; D008807:Mice, Inbred BALB C
D000911:Antibodies, Monoclonal; D008050:Lipid A; D008070:Lipopolysaccharides; D003165:Complement System Proteins