The flocculation of botulinus toxin antitoxin mixtures.

Abstract

Neutral or nearly neutral mixtures of diphtheria toxin and antitoxin give rise to a flocculent precipitate, appearing earliest in those tubes approaching most nearly the point of toxin neutralization. This phenomenon was adapted by Ramon 1 to the in vitro titration of antitoxin, a method the value of which has since been confirmed by a number of investigators.

In the course of an attempt to develop an early specific reaction for the diagnosis of botulism it was hoped that the precipitin test might be useful to detect small quantities of toxin in the circulating blood of laboratory animals suffering from this disease. For the production of the precipitating serum, rabbits were immunized with the formalinized filtrates of four day old botulinus cultures. Before the addition of formalin these filtrates contained approximately 100,000 guinea pig MLD per cc.

The sera obtained were titrated in mice, and 0.1 cc. was found to protect against 6,000 fatal doses. These sera, however, were not flocculated by their homologous toxin. Toxins of various age and potency, and at various pH all gave negative results. A sample of botulinus antitoxin received from the New York City Department of Health gave only a weak and irregular reaction; 0.1 cc. of this serum protected a mouse against 10,000 fatal doses. Under parallel conditions the diphtheria toxin antitoxin mixtures gave typical quantitative precipitations.

We had previously observed that precipitation takes place when the antitoxin is combined with the extracts of foods infected with B. botulinus, 2 as well as with bacterial autolysates, although these reactions were found to be not strictly type specific 3 . In the light of these observations the negative results obtained above led us to suspect that the appearance of the precipitate might be due, not to the actual antitoxin content, but to the presence in the serum of an antibacterial antibody.