DIFFERENTIATION OF THE SEROTYPE-B AND SPECIES-SPECIFIC ANTIGENS OF ACTINOBACILLUS-ACTINOMYCETEMCOMITANS RECOGNIZED BY MONOCLONAL-ANTIBODIES

The serotype b antigens have been reported to be associated with lipop olysaccharide. Using murine monoclonal antibodies specific for either a serotype b antigen or the Actinobacillus actinomycetemcomitans speci es, the relationship of the two epitopes to lipopolysaccharide was det ermined. 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. actinom ycetemcomitans culture supernatants. Serotype b-specific monoclonal an tibodies bound to the polysaccharide of acid-hydrolyzed lipopolysaccha ride. Species-specific monoclonal antibodies bound to both the polysac charide and the lipid A fraction of lipopolysaccharide after acid hydr olysis. Polymyxin b partially inhibited the binding of the species-spe cific monoclonal antibodies to lipopolysaccharide and had no effect on the binding of the serotype b-specific monoclonal antibodies to lipop olysaccharide. Lipopolysaccharide from whole bacteria and polysacchari de material isolated from culture supernatants were separated by gel f iltration 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 pol ysaccharides identical to the blot pattern of lipopolysaccharide molec ules separated by polyacrylamide gel electrophoresis and stained with silver stain. Chemical analysis of the polysaccharide containing serot ype b antigen revealed 85% ribose, 11% glucose, and no lipid. Chemical content of the species-specific antigenic material revealed a composi tion typical of lipopolysaccharide. Immunoelectron microscopy using th e species- or serotype b-specific monoclonal antibodies confirmed the biochemical and immunological characterization of the two antigens, sh owing that the species-specific epitopes were on the surface of the A. actinomycetemcomitans cell membrane and the serotype b-specific epito pes on the amorphous material extending from the cell surface. The dat a 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 molecula r species.