The gene (spaP) coding for the Streptococcus mutans major surface protein antigen P1 (or IIH) has been cloned into Escherichia coli (

A panel of 15 murine monoclonal antibodies (MAbs; 14 immunoglobulin Gl, 1 immunoglobulin G2a) directed against antigen P1, a major surface protein of mutans streptococci, was prepared. All of these MAbs reacted by the enzyme-linked immunosorbent assay with solubilized wall material from Streptococcus mutans Ingbritt 175 (a serotype c strain which retains significant amounts of P1 in its cell wall), culture supernatant fluid from Ingbritt 162 (a strain which excretes large amounts of P1 into the culture medium), and purified P1. By Western immunoblotting, these MAbs were observed to react with a high-molecular-weight polypeptide which comigrated with antigen P1. None of these MAbs cross-reacted with human heart tissue or with various eucaryotic proteins. When whole cells of various strains of mutans streptococci were screened against the panel of MAbs, the strongest reactivities were noted with strains of serotype c and e S. mutans, while a serotype f strain of S. mutans, along with S. sobrinus and S. cricetus strains, reacted somewhat more weakly. S. rattus strains were completely negative. Results obtained with bacterial culture supernatants were qualitatively similar. The surface localization of antigen P1 was confirmed by electron microscopy with an indirect immunogold technique. In sectioned S. mutans cells, labeling appeared to be associated with a fibrillar "fuzzy coat" layer, which was far more prominent on cells of Ingbritt 175 than on those of Ingbritt 162.

Cell membranes of Streptococcus mutans BHT serotype b were prepared after glass bead disruption or mutanolysin digestion of whole cells. Immunoblot analyses of BHT membrane extracts revealed major polypeptides of 42,000, 46,000, 62,000, and 82,000 daltons, as well as several minor bands, to be reactive with rabbit anti-human heart immunoglobulins. Heart cross-reactive antigens have been reported in the cell walls and culture fluids of several S. mutans serotypes. This represents the first report of cell membrane-localized heart cross-reactive antigens in this oral pathogen. Positive enzyme-linked immunosorbent assay and immunoblot reactions were also obtained with heart tissue antigen and anti-BHT sera, indicating mutual cross-reactivity. The major cross-reactive component detected by immunoblotting of human heart extracts was a 69,000-dalton polypeptide.

Monoclonal antibodies (MAb) raised to intact Streptococcus mutans P-4 cells (serotype e) were used to demonstrate the presence of shared antigenic determinant(s) between S. mutans BHT (serotype b) cell membranes and human heart tissue. MAb binding to both BHT membrane and human heart tissue was demonstrated by ELISA. Common antigens were identified by immunoblot analysis following separation of BHT membrane components and human heart antigens by SDS-PAGE. MAb 22C4 recognized three polypeptides from the BHT membrane preparation, having molecular masses of 42, 56 and 85 kDa. MAb 22C4 also recognized an 85 kDa component and a 200 kDa component from human heart tissue. MAb D159 was specific for a single 82 kDa polypeptide in BHT membrane, and also bound to two high molecular mass components in human heart (165 and 200 kDa). When both MAb D159 and 22C4 were first absorbed with S. mutans P-4 cells, subsequent reactivity to the aforementioned BHT membrane components was inhibited, indicating that these cross-reactive components are found in S. mutans P-4 as well as in S. mutans BHT micro-organisms. Competitive binding analysis showed that both MAb D159 and MAb 22C4 bound to myosin, indicating that S. mutans BHT membrane, human heart tissue and myosin share at least one immunodeterminant. This indicates that myosin could be the cross-reactive tissue component in human heart.

Monoclonal antibodies (mAb) generated by immunization of mice with membranes of Streptococcus pyogenes Manfredo (M type 5) were tested for their reactivity with sodium dodecyl sulfate extracts of Streptococcus mutans GS5, Streptococcus rattus BHT (formerly S. mutans, serotype b), and S. mutans Ingbritt 175 in the Western blot. The reaction of the sodium dodecyl sulfate-extracted proteins of whole S. mutans, serotype b), and S. rattus with the mAb revealed that the shared cross-reactive antigens were near m.w. 62,000 to 67,000. Several higher m.w. proteins in S. mutans Ingbritt 175 reacted with the mAb but were not observed in the other two strains of streptococci. Cytoplasmic membranes of S. rattus BHT reacted with these mAb in the enzyme-linked immunosorbent assay and Western blot. The most reactive BHT membrane component detected by these mAb was a 62-kDa polypeptide that was similar in m.w. to the membrane component recognized by these antibodies in purified S. pyogenes membranes. The reactivity of the mAb with BHT membranes in the enzyme-linked immunosorbent assay was absorbed by rabbit skeletal muscle myosin. The patterns of reactivity observed in the BHT membrane immunoblots in this study closely resemble those previously obtained using polyclonal rabbit anti-human heart sera.