Differentiation of the serotype b and species‐specific antigens of Actinobacillus actinomycetemcomitans recognized by monoclonal antibodies

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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.

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