Purification of Vibrio cholerae soluble hemagglutinin and development of enzyme-linked immunosorbent assays for antigen and antibody quantitations

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Abstract
Soluble hemagglutinin (HA) from an El Tor V. cholerae strain (serotype Ogawa) was purified by a sequence of salt precipitation, gel filtration and agarose electrophoresis. The purified material, which gave a single precipitation line in immunodiffusion tests with homologous antiserum, showed immunological identity reactions in double diffusion-in-gel with soluble HA produced by various classical and El Tor strains of different serotypes. Purified HA was used for development of an enzyme-linked immunosorbent assay for titration of specific antibodies against soluble HA and for quantitation of this antigen. Rabbit anti-HA serum reacted in high titer with the soluble HA coated on polystyrene microtiter plates; antiserum against cholera toxin, lipopolysaccharide or whole washed V. cholerae showed little or no reactivity. In inhibition tests, as little as 2.5 ng of soluble HA was detected with the enzyme-linked immunosorbent assay. Culture supernatants of different El Tor and classical V. cholerae strains all completely inhibited the binding of anti-HA antibody to solid-phase-bound homologous antigen; the amounts of HA produced by individual strains varied at least 1000-fold. Only 2 of 10 paired acute- and convalescent-phase sera from Bangladeshi cholera patients showed significant titer increases against soluble HA in parallel titrations.