The study of intestinal immunity against V. cholerae: purification of V. cholerae El Tor haemagglutinin and the protective role of its antibody in experimental cholera.

ABSTRACT

V. cholerae El Tor Ogawa strain O17SR grown on trypticase soy agar were extracted with 0.05 M cyclohexylamino propane sulfonic acid (CAPS) pH 9.5 at 37 degrees C for 1 hour. The bacteria were then removed by centrifugation and millipore filtration. The filtered fluid, after being dialysed against many changes of cold distilled water, was concentrated and passed through Sephadex G200 column. Three protein profiles were eluted out with 0.05 M Tris buffer pH 8.6. The haemagglutinin and the bacterial lipopolysaccharide (LPS) were confined to the first profile. They were subsequently separated by agarose gel electrophoresis. The haemagglutinin was found to be more anodic than the LPS. After homogenization of the gel strips containing the haemagglutinin followed by centrifugation at 9,000 g pure haemagglutinin was obtained in the supernatant. Rabbit aniserum against pure haemagglutinin contained protective antibodies against V. cholerae infection in the baby mouse model. Specific antibodies prepared from this antiserum was as protective as the antibodies directed against whole V. cholerae and heat stable somatic antigens of V. cholerae upon the same weight unit.