Development of a Purified Cholera Toxoid I. Purification of Toxin

Abstract

The enterotoxin from Vibrio cholerae is selectively concentrated from cell-free culture supernatant by co-precipitation with hexametaphosphate and is further purified by adsorption on aluminum hydroxide powder. The bulk of residual somatic antigen becomes insoluble upon lyophilization of the toxin preparation and is removed by centrifugation of the rehydrated material. Other contaminants are eliminated by treatment with activated carbon. Preparations of toxin, purified by this method, have been characterized by: (i) a single immunoprecipitin line against polyvalent antisera; (ii) homogeneity on acrylamide gels; (iii) specific activities on the order of 22 limit-of-bluing doses/μg; (iv) ultraviolet spectra characteristic of pure protein; and (v) overall yields on the order of 50%, irrespective of purification scale. Such preparations, however, have been shown to contain trace amounts of somatic antigen when they are intensively tested either for their ability to elevate serum vibriocidal antibody titers in immunized rabbits or for their ability to increase resistance of immunized mice to live vibrio challenge. In the latter test system, the level of residual somatic antigen per 50 μg of toxin (toxoid) antigen generally did not exceed 0.025% of the Division of Biological Standards reference vaccine, V. cholerae Inaba IN-12. Methods for elimination of this small amount of somatic antigen have been investigated and are discussed. The particular combination of purification steps which are presently described have been easily and reproducibly applied on a production scale to prepare gram amounts of toxin with a high degree of purity, even under a variety of initial conditions.