Monoclonal antibodies (MAbs) were prepared against the B. loescheii adhesins responsible for this organism's coaggregation with Streptococcus sanguis 34 and Actinomyces israelii PK14 by screening hybridoma supernatants specifically for coaggregation- blocking activity. Ten hybridomas were selected for preparation of ascites fluid in mice. Of these, 4 specifically blocked the B. loescheii - S. sanguis coaggregation and 2 inhibited the B. loescheii - A. israelii interaction. MAb or Fab' fragments inhibited these coaggregations at low levels suggesting that the antibodies were specific for regions near or at the binding sties of the respective adhesins. These MAbs were used to confirm that the adhesins consisted of a 75kD (S. sanguis specific adhesin) and 45kD (A. israelii specific adhesin) subunits, respectively, using radiolabeled adhesin-containing preparations. In the native form, the S. sanguis adhesin is a pentamer with a M.W. of 380kD. Using radiolabeled MAbs in direct binding assays, it was determined that each B. loescheii cell carries a maximum of approximately 300 S. sanguis specific adhesins and 600 A. israelii specific adhesins on its surface. The MAbs, were used also to localize the respective adhesins on the surface of the cells. Both types of adhesins were shown to be associated with the distal portion of the organisms fimbriae. The adhesions were arranged randomly in small to large clusters which extended away from the fimbriae. Preliminary studies with the radiolabeled adhesions suggest that the two types of adhesions are closely associated with one another on the surface of the cell. Employing the same strategy described above, MAbs specific for the afimbriate 250kD adhesin of Capnocytophaga gingivalis, which mediates its coaggregation with A. israelii PK16, are currently being prepared. Concurrently, the carbohydrate receptor of S. sanguis 34 which mediates coaggregation with Capnocytophaga ochracea is being purified and characterized.
