title: Differentiation of the serotype b and species-specific antigens of Actinobacillus actinobacillus actinomycetemcomitans recognized by monoclonal antibodies.
abstract: The serotype b antigens have been reported to be associated with lipopolysaccharide. Using murine monoclonal antibodies specific for either a serotype b antigen or the Actinobacillus actinomycetemcomitans species, the relationship of the two epitopes to lipopolysaccharide was determined. Both the species-specific and serotype b-specific monoclonal antibodies bound to whole cells, vesicles and conventionally isolated lipopolysaccharide and polysaccharide material derived from A. actinomycetemcomitans culture supernatants. Serotype b-specific monoclonal antibodies bound to the polysaccharide of acid-hydrolyzed lipopolysaccharide. Species-specific monoclonal antibodies bound to both the polysaccharide and the lipid A fraction of lipopolysaccharide after acid hydrolysis. Polymyxin b partially inhibited the binding of the species-specific monoclonal antibodies to lipopolysaccharide and had no effect on the binding of the serotype b-specific monoclonal antibodies to lipopolysaccharide. Lipopolysaccharide from whole bacteria and polysaccharide material isolated from culture supernatants were separated by gel filtration chromatography in deoxycholate into fractions that contained serotype b antigen, both serotype b and species-specific antigens, or species-specific antigen. SDS-polyacrylamide gel electrophoresis and Western blotting analysis of the fractions revealed that the serotype b antigen was on a high-molecular-weight polysaccharide material. The species-specific antigen was on a ladder of lower-molecular-weight polysaccharides identical to the blot pattern of lipopolysaccharide molecules separated by polyacrylamide gel electrophoresis and stained with silver stain. Chemical analysis of the polysaccharide containing serotype b antigen revealed 85% ribose, 11% glucose, and no lipid. Chemical content of the species-specific antigenic material revealed a composition typical of lipopolysaccharide. Immunoelectron microscopy using the species- or serotype b-specific monoclonal antibodies confirmed the biochemical and immunological characterization of the two antigens, showing that the species-specific epitopes were on the surface of the A. actinomycetemcomitans cell membrane and the serotype b-specific epitopes on the amorphous material extending from the cell surface. The data indicated that the serotype b antigen, detected by the antibody, was separable from lipopolysaccharide and was an A. actinomycetemcomitans capsular material. The species-specific antigen, being more conserved than the serotype antigen, was on all the lipopolysaccharide molecular species.
authors.affiliation: Department of Oral Biology, College of Dentistry, University of Florida, Gainesville, USA.
authors.forename: W P
authors.initials: WP
authors.lastname: McArthur
authors.forename: S
authors.initials: S
authors.lastname: Stroup
authors.forename: S
authors.initials: S
authors.lastname: McClellan
authors.forename: K P
authors.initials: KP
authors.lastname: Leung
mesh_terms: D016976:Aggregatibacter actinomycetemcomitans / Q000145:classification / Q000276:immunology*; D000818:Animals; D000911:Antibodies, Monoclonal; D000942:Antigens, Bacterial / Q000737:chemistry / Q000276:immunology*; D000954:Antigens, Surface / Q000737:chemistry / Q000276:immunology*; D016667:Bacterial Capsules / Q000737:chemistry / Q000276:immunology; D001425:Bacterial Outer Membrane Proteins / Q000276:immunology; D015153:Blotting, Western; D000939:Epitopes; D008070:Lipopolysaccharides / Q000276:immunology; D051379:Mice; D011135:Polysaccharides, Bacterial / Q000032:analysis / Q000276:immunology*; D011485:Protein Binding; D012703:Serotyping; D013045:Species Specificity
chemical_list: D000911:Antibodies, Monoclonal; D000942:Antigens, Bacterial; D000954:Antigens, Surface; D001425:Bacterial Outer Membrane Proteins; D000939:Epitopes; D008070:Lipopolysaccharides; D011135:Polysaccharides, Bacterial