Isolation and characterization of murine monoclonal antibodies specific for gram-negative bacterial lipopolysaccharide: association of cross-genus reactivity with lipid A specificity

Abstract

Somatic cell hybrids secreting monoclonal antibodies against the core-glycolipid portion of enterobacterial endotoxin were derived from mice immunized with Escherichia coli J5 or Salmonella minnesota R595 heat-killed organisms or lipopolysaccharide (LPS). Eight antibodies were selected for their ability to cross-react with several members of a panel of gram-negative bacterial antigens in a radioimmunoassay. This panel represented five genera and two families of organisms: E. coli O111:B4, E. coli O55:B5, Klebsiella pneumoniae, Pseudomonas aeruginosa, Salmonella minnesota, and Serratia marcescens. The binding sites for six of the antibodies were unequivocally localized within the lipid A moiety of the endotoxin molecule by using the radioimmunoassay on LPS and free lipid A. The anti-lipid A antibodies were further characterized for their ability to interact with LPS variants by using a sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunostaining procedure. The monoclonal antibodies bound almost exclusively to the low-molecular-weight species of LPS on the polyacrylamide gel. These components corresponded to LPS isolated from rough strains of organisms (strains which lack O-specific carbohydrate). These results suggested that the cross-reactive component of antisera raised against rough mutants of gram-negative bacteria contain antibodies of lipid A specificity. Moreover, the determinant within the lipid A moiety of LPS may have been accessible to the monoclonal antibodies only in those endotoxin molecules on the outer membrane surface which lack the O-specific carbohydrate.