Date of Award

1987

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Abstract

Vibrio cholerae is typed serologically on the basis of somatic O-antigens. The serovar-specific antigens A, B, and C of V. cholerae O1 are located in the polysaccharide (PS) moiety of the lipopolysaccharide (LPS) in the cell wall, but the exact structure and composition of these antigens are unknown. Presently, V. cholerae non-O1 serovars are differentiated serologically by two independent typing systems. Each system utilizes whole cell vaccines which result in typing sera containing serovar-specific, anti-PS activity and non-specific, cross-reacting activities. Serovar-specific anti-PS sera were produced in rabbits immunized with PS extracted from selected vaccine strains of V. cholerae O1 and non-O1 conjugated with a protein carrier. In the passive hemagglutination assay versus LPS coated rabbit erythrocytes, the PS sera displayed titers comparable to those obtained with antisera from rabbits immunized with whole cell vaccines. In the slide agglutination test, anti-PS sera serologically distinguished isolates which had previously agglutinated in more than one typing serum. Thus, serovar-specific PS-associated antigens of V. cholerae are discernible serologically, in vitro, and excellent immunogens when conjugated to a protein carrier, in vivo. In addition, hybridomas secreting monoclonal antibodies (MAbs) directed against V. cholerae O1 were prepared by fusing spleen cells from mice immunized with whole cell vaccines and SP2/O-Ag14 myeloma cells. A library of nineteen MAbs was established; seven were specific for the A antigen, two for antigen B, and one for antigen C, as determined in an enzyme linked immunosorbent assay (ELISA) utilizing seven O1 and nine non-O1 LPS preparations as the antigens. Each MAb displayed specificity when tested by slide agglutination and by ELISA against whole cell preparations of 36 O1 and 52 non-O1 V. cholerae serovars, 20 heterologous Vibrio species, and 37 heterologous bacterial species. Three distinct reactivity patterns were exhibited among the anti-A clones. The reactivity and specificity of each MAb were further evaluated by the direct immunofluorescence assay and by immunoelectron microscopy on thin sectioned and whole cell mounts of V. cholerae O1. Finally, a mixture of MAbs increased the sensitivity of the reagent.

Pages

113

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