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A Quantitative Study of the Ramon Diphtheria Flocculation Reaction

From the Antitoxin and Vaccine Laboratory of the Massachusetts Department of Public Health, Jamaica Plain, Massachusetts

Abstract

1. An absolute quantitative method for the titration of diphtheria toxin and antitoxin, similar to that used by Heidelberger and his co-workers for the precipitin reaction, has been described.

2. The results demonstrate once more that toxin and antitoxin unite in more than one proportion. An explanation of the Danysz phenomenon is given in these terms.

3. Estimated values for the amount of toxin nitrogen per Lf unit, obtained from six titrations, varied between 0.00042 and 0.00048 mgm. nitrogen per Lf, regardless of the purity of the toxin and antitoxin preparations used.

4. The maximum antitoxic antibody nitrogen precipitable from 300 units of antitoxin, either as whole sera or ammonium sulfate concentrates, is approximately 0.47 or 0.0016 mgm. per unit. One highly opalescent concentrated antitoxin gave a somewhat higher figure and a sample of concentrated and purified "globulin-modified" antitoxin from the Lederle Laboratories gave a lower figure.

5. The equivalence or neutral zone lies between 0.5 and 1.4 Lf units of toxin per unit of antitoxin. The ratio of antitoxin nitrogen to toxin nitrogen is about 7.0 at the antitoxin end of the neutral zone, about 3.5 at the flocculation point and about 2.5 in the region of toxin excess.

6. The flocculation reaction is closely analogous to the precipitin reaction but differs from it in that flocculation occurs only in the immediate neighborhood of the equivalence zone.

7. The results calculated from these experiments are not expected to undergo significant change even though purer toxins and antitoxins may be used, provided that the purification process does not cause a splitting or degradation of the toxin or antitoxin molecules.

Footnotes

¹ Edward Hickling Bradford Fellow, Harvard Medical School.