Comparative Studies of Infusion and Infusion-Free Diphtheria Toxin in Antitoxin Production and in Standardization by the Flocculation, Subcutaneous, and Intracutaneous Tests
Authors: Kirkbride Mary B. , Berthelsen Knud C. , Clark Rose F. 
Published: January 2023

Summary
The practical application to routine and experimental purposes of diphtheria toxin produced with an infusion-free peptone medium is reported. The procedures followed in the routine preparation of the medium and toxin are given in some detail. The marked reduction in cost of production of the toxin as compared with that of meat-infusion toxin, and the greater uniformity in the potency obtained are pointed out.
Results of comparative studies of the flocculation-reaction test are recorded. The infusion-free toxin was found to be at least as satisfactory as the infusion toxin, in these tests. The micrometer-syringe method proved less accurate than the “pipette method” in which larger volumes of toxin are measured for the test.
A comparison of the flocculation-reaction test with the subcutaneous and the intracutaneous animal method for determining diphtheria antitoxic values showed closer agreement with the subcutaneous test. Variation was less than 10 per cent with the subcutaneous and nearly 20 per cent with the intracutaneous method. In titrations of sera from certain horses, especially during the early stages of immunization, wide variations were noted between the results of the flocculation and subcutaneous tests on the one hand and those of the intracutaneous test on the other. The flocculation reaction was found to be a satisfactory method for the approximate estimation of the unit content of concentrated antitoxins. The flocculation test was used to determine the time at which optimum antitoxic titer might be expected in the serum of horses immunized against the infusion-free toxin by different methods.
The value of the toxin as an antigen was also studied in a series of twelve horses. The results demonstrated that the infusion-free toxin is equal to, if not better than, meat-infusion toxins in the routine immunization of horses for diphtheria antitoxin production. Three horses given injections of purified (ultrafiltered) infusion-free peptone toxin appeared to respond as well as horses receiving other toxins and possibly with less disturbance of the normal metabolic processes. Further studies are in progress.
The chief points which are brought out by this investigation may be enumerated as follows: Diphtheria toxin of uniformly high potency has been produced in infusion-free peptone medium on a routine basis for a period of nearly two years.The infusion-free toxin compares in all respects very favorably with routine infusion toxin for the immunization of horses in the production of diphtheria antitoxin.This toxin has also been used extensively in comparative flocculation-reaction tests and found in all respects to compare favorably with the infusion toxin.The flocculation-reaction test for standardization of diphtheria antitoxin proved more accurate when relatively large volumes were measured by pipettes than when the micrometer-syringe method was used.Closer agreement was found between the flocculation-reaction-test and the subcutaneous animal method for the determination of antitoxic content than between the flocculation and the intracutaneous animal test, or between the subcutaneous and the intracutaneous test.The flocculation-reaction test was satisfactory for preliminary estimations of the antitoxin content of the concentrated as well as of the untreated serum.Besides its other advantages, the great saving in production cost as compared with the usual meat-infusion toxin makes the infusion-free peptone toxin an important economic factor where large amounts of toxin are required for the production of diphtheria antitoxin, for standardization, and possibly also, for the active immunization of persons with toxoid.