Characterization of monoclonal antibodies specific for adhesion: isolation of an adhesin of Streptococcus sanguis FW213.
Monoclonal antibodies reactive to an adhesive strain of Streptococcus sanguis (FW213) and nonreactive to a nonadhesive mutant (JL7) were derived from the fusion of myeloma line X63Ag8.653 and spleen cells from BALB/c mice immunized with live S. sanguis cells. Five cell lines, belonging to subclasses of immunoglobulin G, produced monoclonal antibodies specifically directed against the adhesive strain. All five antibodies also failed to react with five additional, independently isolated, nonadhesive mutants. A spontaneous mutant of FW213 (VT508) that no longer reacted with monoclonal antibody F51 (MAbF51) was isolated by serial agglutination with the antibody. Langmuir adsorption isotherms of VT508 indicated that this mutant also had altered ability to adhere to saliva-coated hydroxyapatite further confirming the specificity of MAbF51 for adhesion. Electron microscopy revealed that VT508 had lost the peritrichous fimbriae associated with the adhesion of FW213. MAbF51 was used to purify the adhesin from lysozyme cell extracts by using an affinity column of MAbF51 linked to Sephacryl S1000. Purity was suggested by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and crossed immunoelectrophoresis (CIEP). The adhesin had a molecular weight greater than 150,000 and was not denatured in sodium dodecyl sulfate reducing gels. Two peaks of near electrophoretic mobility were detected in CIEP when the purified material was run against polyclonal antibody to the whole cell. Tandem CIEP analysis and immunoprecipitation provided evidence that the two peaks represented the same antigen in two different forms.
D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology*; D000936:Antigen-Antibody Complex; D000942:Antigens, Bacterial / Q000302:isolation & purification*; D001422:Bacterial Adhesion; D004797:Enzyme-Linked Immunosorbent Assay; D007123:Immunoelectrophoresis, Two-Dimensional; D051379:Mice; D008807:Mice, Inbred BALB C; D008854:Microscopy, Electron; D008970:Molecular Weight; D009154:Mutation; D013045:Species Specificity; D013298:Streptococcus sanguis / Q000235:genetics / Q000502:physiology* / Q000648:ultrastructure
D000911:Antibodies, Monoclonal; D000936:Antigen-Antibody Complex; D000942:Antigens, Bacterial