Diphtheria toxin-antitoxin titration by Ramon method for practical application.

The Ramon test was evolved from the works of Calmette and Massol, 1 who in 1909 applied the flocculation test for titration in vitro of antivenom serum against Cobra venom. Nicolle, Cesari and Debains 2 in 1919 applied the same principle for titration of diphtheria and tetanus toxin-antitoxin by the method of Ascoli. 3 The reaction consisted in the formation of an opalescent ring in contact with a concentrated toxin and gelatinized antitoxin. Georgi 4 added a suspension of cholesterolized heart extract to the mixtures of toxin-antitoxin to obtain flocculation.

Ramon 5 in 1922 found that diphtheria and tetanus toxin and antitoxin alone, when mixed in certain proportions, will bring about flocculation. The mixtures with a deficiency or excess of either toxin or antitoxin will fail to flocculate. The first precipitate to appear in the mixtures Ramon calls the “precipitate indicateur” and this corresponds to a nearly neutral mixture. From the latter the values of toxin or antitoxin are calculated.

Scholtz 6 in 1923 found that it was noit necessary to employ for this test the enormous quantities of toxin used by Ramon (20 cc. for each tube). He used instead 2 cc. of a stable titrated toxin and corresponding diiutions of serum. Glenny and Okell 7 in 1924 also used from 2 to 5 cc. of a stable titrated toxin and minute quantities of undiluted serum. A11 workers agree that the Ramon titrations agree closely with guinea pig tests and are therefore applicable for practical routine estimation of values of toxin and antitoxin.

Povitzky and Banzhaf working on this test since early spring of 1924 arrived at the same conclusions. They found that the most: reliable and constant results are obtained by the use of a potent stable toxin titrated for its L+ and flocculation values and undiluted serum or (if the latter is sufficiently strong) diluted 1 :I, that is, with an equal amount of physiological salt solution. The amount of toxin was usually 2.5 cc. per tube.