Monoclonal antibody activity against native and denatured forms of gonococcal outer membrane proteins as detected within ultrathin, longitudinal slices of polyacrylamide gels.
Monoclonal antibodies were used to analyze the antigenic properties of denatured and native forms of gonococcal outer membrane proteins. The protein samples were only partially dissociated by treatment for 30 min at 40 degrees C with 0.1% (w/v) SDS, 0.5% (v/v) Triton X-100, and then processed by polyacrylamide gel electrophoresis without boiling. The resulting pattern included the native aggregated and trimeric forms of protein I and III as they exist in the gonococcal outer membrane, as well as the denatured monomeric forms. Two methods were compared to analyze these gels: gel immunoradioassay (GIRA), and Western blotting. With GIRA longitudinal 50 micron thin slices, up to 40 identical copies per gel, were produced with a microtome cryostat. These slices were exposed to the monoclonal antibody and antibody binding was detected by 125I-protein A and autoradiography. Serotype-specific, monoclonal antibodies reacted most commonly with the native polymeric form of gonococcal protein I and less frequently recognized the denatured, monomeric form. Monoclonal antibodies that recognized the polymeric form of protein I frequently produced antibody-mediated, complement-dependent, bactericidal activity for gonococci bearing the same protein I serotype. The antigen specificity of these functionally relevant antibodies could not be characterized by the Western blotting procedure, which produced incomplete transfer to nitrocellulose paper of the polymeric, high molecular weight protein aggregates. A third technique, radioimmunoprecipitation using partial dissociating conditions, did not permit differentiation between proteins I- and III-specific monoclonals after analysis of the precipitated material by denaturing SDS electrophoresis.
D000818:Animals; D000911:Antibodies, Monoclonal; D001425:Bacterial Outer Membrane Proteins / Q000032:analysis* / Q000276:immunology; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods; D004797:Enzyme-Linked Immunosorbent Assay; D000939:Epitopes / Q000032:analysis*; D051379:Mice; D008807:Mice, Inbred BALB C; D009344:Neisseria gonorrhoeae / Q000032:analysis*; D011489:Protein Denaturation; D011863:Radioimmunoassay / Q000379:methods
D000911:Antibodies, Monoclonal; D001425:Bacterial Outer Membrane Proteins; D000939:Epitopes