Production and characterisation of monoclonal antibodies to outer-membrane-protein antigens of Vibrio cholerae O1.
Monoclonal antibodies (MAbs) were raised against the major, 46-48-Kda outer-membrane proteins of Vibrio cholerae O1. The hybridoma clones were screened by enzyme-linked immunosorbent assay (ELISA) with cell-surface proteins of V. cholerae O1 as the coating antigen. Four hybridomas, which secreted anti-V. cholerae cell-surface-protein antibodies, were subcloned by limiting dilution and obtained as ascites in vivo. A MAb of the IgG1 subclass was isolated in good yield from the murine ascites by affinity chromatography with recombinant protein G-Sepharose 4B. It gave positive reactions, as determined by ELISA, against cell-surface proteins prepared from both biotypes (classical and El Tor) and both serotypes (Ogawa and Inaba) of V. cholerae O1. The MAb did not have any reactivity towards V. cholerae lipopolysaccharide preparations. Immunoblotting studies were performed on cell-surface proteins separated by one-dimensional sodium dodecyl sulphate-polyacrylamide gel electrophoresis (1D SDS-PAGE) and also by two-dimensional (2D) electrophoresis with iso-electric focusing in the first dimension followed by SDS-PAGE in the second dimension. When proteins were separated by 1D SDS-PAGE, only one band at 46-48 Kda reacted with the MAb. This protein appeared to consist of two narrowly-spaced and cross-reactive bands when a nitrocellulose blot, obtained by 2D SDS-PAGE, was exposed to the MAb.
Kabir
S
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Department of Immunology, Karolinska Institute, Stockholm, Sweden.
D000818:Animals; D000911:Antibodies, Monoclonal / Q000096:biosynthesis*; D000942:Antigens, Bacterial / Q000276:immunology*; D000954:Antigens, Surface / Q000276:immunology; D001425:Bacterial Outer Membrane Proteins / Q000276:immunology*; D002846:Chromatography, Affinity; D003429:Cross Reactions; D004492:Edetic Acid; D015180:Electrophoresis, Gel, Two-Dimensional; D004591:Electrophoresis, Polyacrylamide Gel; D004797:Enzyme-Linked Immunosorbent Assay; D006825:Hybridomas; D051379:Mice; D008807:Mice, Inbred BALB C; D014734:Vibrio cholerae / Q000276:immunology* / Q000302:isolation & purification
D000911:Antibodies, Monoclonal; D000942:Antigens, Bacterial; D000954:Antigens, Surface; D001425:Bacterial Outer Membrane Proteins; D004492:Edetic Acid