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.
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.
Rozalski
A
A
Division of Biochemical Microbiology, Research Centre Borstel, Centre for Medicine and Biosciences, Germany.
Brade
L
L
Kosma
P
P
Moxon
R
R
Kusumoto
S
S
Brade
H
H
D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology* / Q000502:physiology; D001667:Binding, Competitive / Q000276:immunology; D015153:Blotting, Western; D000939:Epitopes / Q000737:chemistry / Q000276:immunology*; D019085:Fluorescent Antibody Technique, Direct; D006023:Glycoproteins / Q000276:immunology*; D006193:Haemophilus influenzae / Q000235:genetics* / Q000276:immunology; D008070:Lipopolysaccharides / Q000276:immunology*; D051379:Mice; D008807:Mice, Inbred BALB C; D009154:Mutation / Q000276:immunology / Q000502:physiology; D009693:Nucleic Acid Hybridization; D010750:Phosphoproteins / Q000276:immunology*
D000911:Antibodies, Monoclonal; D000939:Epitopes; D006023:Glycoproteins; D008070:Lipopolysaccharides; D010750:Phosphoproteins