Characterization of monoclonal antibodies recognizing three distinct, phosphorylated carbohydrate epitopes in the lipopolysaccharide of the deep rough mutant I-69 Rd−/b+ of Haemophilus influenzae |
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Authors: | Antoni Rozalski,Lore Brade,Paul Kosma,Richard Moxon,Shoichi Kusumoto,& Helmut Brade |
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Affiliation: | Division of Biochemical Microbiology, Research Centre Borstel, Centre for Medicine and Biosciences, D-23845 Borstel, Germany.; Institute of Chemistry, University of Agriculture, A-1190 Vienna, Austria.; Institute of Molecular Medicine, John Radcliffe Hospital, Headington, Oxford OX3 9DU, UK.; Department of Chemistry, Faculty of Science, Osaka University, Toyonaka, Osaka 560, Japan. |
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Abstract: | Monoclonal antibodies against the lipopolysaccharide (LPS) of the deep rough mutant I-69 Rd−/b+ of Haemophilus influenzae were obtained after immunization of mice with sheep erythrocytes which had been coated with de- O -acylated LPS. Characterization of antibodies was performed by enzyme immuno assay (EIA) using LPS or neoglycoconjugates containing partial structures of LPS as solid-phase antigens and by haemagglutination with sheep erythrocytes coated with de- O -acylated LPS. Binding data were confirmed by EIA inhibition experiments using deacylated LPS or synthetic partial structures thereof. Three antibodies were specific for 3-deoxy- d - manno -octulopyranosonic acid- (Kdo) 5-phosphate, one for Kdo-4-phosphate, and one required, in addition to a Kdo-phosphate, parts of the phosphorylated glucosamine backbone of lipid A. All antibodies also bound in (i) Western blots to bacterial whole-cell lysates or isolated LPS separated by SDS–PAGE, (ii) bacterial colony blots, and (iii) immunofluorescence with live bacteria. The latter result indicated that Kdo-4- and Kdo-5-phosphate are synthesized by the bacteria and are not the result of phosphate migration. |
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