ABSTRACT
Eleven monoclonal antibodies were produced using whole Bordetella pertussis cells as the immunizing antigen. All monoclonal antibodies reacted with components of Bordetella pertussis, as visualized in immunoblotting of SDS polyacrylamide gels. Selected antibodies were coupled to Sepharose columns and used for isolation of the corresponding antigen. In all cases complete accordance was found between SDS polyacrylamide gel electrophoresis of the eluted antigen and the bands found in immunoblotting of the original extract stained with the respective monoclonal antibody. One major problem in the interpretation of the results was the finding that some of the monoclonal antibodies stained a number of bands in immunoblottings of crude B.pertussis extract. This phenomenon was shown to be caused by proteolytic degradation of the antigens, since prior addition of protease inhibitors to the extract resulted in the staining of only one band. The monoclonal antibodies showed different reactivity patterns with various strains of B.pertussis, B.parapertussis, B.bronchiseptica and less closely related bacteria. Two of the antibodies were strictly specific for B.pertussis.
Subject(s)
Antibodies, Monoclonal/immunology , Antigens, Bacterial/isolation & purification , Bordetella pertussis/immunology , Bacteriological Techniques , Chromatography, Affinity , Electrophoresis, Polyacrylamide Gel , Enzyme-Linked Immunosorbent Assay , Hybridomas , Species SpecificityABSTRACT
Two monoclonal antibodies against filamentous haemagglutinin (F-HA) from Bordetella have been produced. In immunoblotting of a SDS-polyacrylamide gel electrophoresis (SDS-PAGE) of high ionic strength extracts of Bordetella pertussis, the two monoclonal antibodies both stained several high molecular weight bands. The two monoclonal antibodies were used for immuno-affinity column one-step purification of F-HA from high-ionic-strength extracts of Bordetella pertussis. In gradient SDS-PAGE, the eluted F-HA is present as a major double band with a molecular weight of approximately 240,000 daltons and several minor bands with molecular weights down to 90,000 daltons. The monoclonal antibodies were used in a monoclonal antibody catching enzyme linked immunosorbent assay (ELISA) for sensitive and specific detection of F-HA. This ELISA system proved valuable in optimizing the elution conditions for the monoclonal affinity columns.
