The Titration of Diphtheria Toxin and Antitoxin by Ramon's Flocculation Method
Authors: Bayne-Jones Stanhope 
Published: January 2023

Summary and Conclusions
In view of the desirability of having a means of titrating diphtheria toxin and antitoxin in the test tube, largely eliminating the use of animals, Ramon's flocculation method has been repeated. In this procedure, toxin and antitoxin are mixed in certain proportions and incubated until flocculation occurs in one of the tubes.
A diphtheria toxin, to be used in the subsequent tests, is first standardized by comparison with an antitoxic serum of definitely known unit value. The amount of toxin which will cause initial flocculation with 1 unit of antitoxin is determined. This amount is given the symbol Lf, and from it is calculated the number of units of antitoxin to which 1 cc. of the toxin is equivalent by flocculation.
The Lf value of the toxin is directly proportional to the original M.L.D. and is equal to the Lo amount when the toxin is fresh. This index of the flocculating power can be used to determine the M.L.D. of a fresh toxin, to follow the course of the production of toxin in a culture, and to find the greatest original toxicity of old or chemically modified toxins. It is independent of the actual toxicity of a changed toxin and hence has no relation to the Lf dose. The Lf value remains constant for a long time after the M.L.D. and L+ dose of the toxin have greatly increased due to reduction of the toxicity produced by age, heat, formaldehyde or other chemicals. The reaction with such toxins is delayed. In all conditions, the Lf value, as noted by Glenny and Okell, is an indication of the capacity of the toxin to combine with antitoxin. It demonstrates qualities of toxin not shown by animal tests and gives valuable information about modified toxins which may be used for the immunization of horses and man.
After the flocculating power, or Lf amount of a toxin has been determined, it serves as a point of reference for the estimation of the value of antitoxins. The method of performing this titration with several slight modifications of Ramon's technic, and the factors influencing the flocculation reaction are described and discussed in this paper. It was possible satisfactorily to use 2 cc. of toxin in each tube, to incubate the tests at 55°C. in order to hasten flocculation, and to deliver small amounts of serum into the tubes by means of the micrometer syringe devised by Trevan.
Old antitoxic sera, the fresh sera of some immunized horses and concentrated pseudoglobulin preparations of diphtheria antitoxin fail to cause flocculation. By mixing these preparations with fresh antitoxic serum, they can be titrated by Ramon's method.
The results obtained by flocculation in the titration of different samples of diphtheria antitoxin in serum, plasma and concentrated pseudoglobulin solutions agreed within about 3 per cent with the values of these preparations as determined by tests on animals.
The method, therefore, was found to be simpler, more rapid, subject to better control and less expensive than the usual way of titrating toxins and antitoxins by the use of animals. It is reliable and accurate.
Ramon's flocculation method is practically useful to those engaged in the preparation and standardization of toxin and antitoxin, and will be a valuable means of advancing the investigation of the nature of toxin and antitoxin and their combinations.