A sensitive and specific reversed passive hemagglutination (RPHA) assay for
cholera enterotoxin has been developed. Equine anti-
choleragenoid antibodies purified by immunoadsorption were covalently coupled to formalinized sheep erythrocytes, using
bis-diazotized benzidine, and the
antitoxin-sensitized erythrocytes were shown to agglutinate specifically in the presence of
cholera enterotoxin. In a microtiter RPHA assay system, the smallest quantity of
enterotoxin that caused hemagglutination was approximately 20 pg. A sensitive assay for
antibodies to
enterotoxin was also developed, based on inhibition of RPHA. Using such assays, we demonstrated that several nontoxinogenic (tox-) strains of Vibrio cholerae produced small but detectable yields of
enterotoxin, 4 to 16 ng/ml, under conditions where the highly toxinogenic strain 569B Inaba produced approximately 16 microgram of
enterotoxin per ml. The
enterotoxin produced in small quantities by these tox- strains was found to be identical to the
enterotoxin from V. cholerae 569B Inaba iv its immunological and
biological activities. Strains of V. cholerae that produce intermediate yields of
enterotoxin have been obtained by two techniques: (i) as less toxinogenic mutants derived from highly toxinogenic strains and (ii) as more toxinogenic mutants derived from tox- strains. Thus, the yield of
enterotoxin in cultures of V. cholerae grown under standardized conditions is is a genetically controlled trait that can be altered by mutation.