Evidence is presented which confirms that
cholera toxoids obtained by reaction of purified toxin with
Formalin possess the ability to partially reactivate both in vivo and in vitro. At the same time, conditions are presented for the preparation of stable, antigenic
cholera toxoids by reaction of purified toxin with
glutaraldehyde. Treatment of purified
cholera toxin with approximately 200 mol of
glutaraldehyde per mol of toxin at pH 7.8 reproducibly resulted in the preparation of
toxoids which: (i) possessed less than 20 bluing doses per 100 mug; (ii) did not reactivate in vivo or in vitro; (iii) precipitated with, and neutralized
antitoxin; (iv) elevated prolonged serum
antitoxin in immunized rabbits; (v) protected immunized guinea pigs against toxin skin challenge; and (vi) lent themselves to enhanced antigenicity by means of an in situ adjuvant system which may be suitable for man. Acrylamide gel electrophoresis and molecular sieve chromatography of a series of
glutaraldehyde-derived
toxoids suggested that the reaction products consisted of monomeric and polymeric species and that the proportion of higher-molecular-weight species was determined by the relative concentrations of toxin and
glutaraldehyde. The results suggested a relationship between complete and irreversible elimination of toxicity and the formation of higher-molecular-weight
toxoids.