EDWIN J. BANZHAF 751 



Several methods have been recommended for standardizing the antigenic (active im- 

 munizing value) of toxoids and toxin-antitoxin mixtures. Park and Banzhaf injected i/io of 

 a human dose subcutaneously into guinea pigs, giving three doses at weekly intervals. Three 

 months later they determined roughly the degree of immunity by injecting intracutaneously 

 the guinea pigs with 1/150, i/ioo, and 1/50 of a fatal dose. A negative reaction with the 

 highest dose was interpreted as a satisfactory product for active immunization. Glenny and 

 his co-workers recommend injecting the guinea pigs with one human dose and three weeks 

 later testing the animals by the Schick method. The products which gave a negative reac- 

 tion were classed as good, active immunizers. Ramon and others rely to a great extent on 

 the antigenic value as shown by the fiocculation test. 



During 1923 when I determined the immunizing value of the products of the New 

 York City laboratory, practice was made of injecting the negatively reacting guinea pigs 

 with toxin. It was found that some of these guinea pigs might fail to survive three fatal doses, 

 whereas others would survive twenty fatal doses, it being understood that the twenty fatal 

 doses were given as the first and only injection of toxin. 



Further observations on those preparations of toxin-antitoxin mixtures that gave a high 

 percentage of active immunization in humans showed that where I mil (one-half the human 

 dose) was injected into guinea pigs, one month later these animals would survive the injec- 

 tion of at least five fatal doses; whereas when the elapsed time was six weeks, the animals 

 would survive at least eight to ten fatal doses. 



In determining the active immunizing value of the toxoids, the guinea pigs are injected 

 with ^ mil (the recommended human dose for the first injection), and one month later five 

 fatal doses of toxin are given. Survival is interpreted as indicating that this product will 

 give a high percentage of actively immunized individuals among those treated. 



The use of toxin-antitoxin mixtures has been criticized on the assumption that 

 the antitoxin in the mixture, which amounts to about 0.000038 of a mil of partially 

 purified antitoxic horse serum to each human dose, would sensitize the individuals 

 to subsequent injections of horse serum. The observations of Hooker and of Park 

 indicate that a skin sensitivity may develop. There has been no convincing evidence 

 that a systemic sensitization ever develops which would lead to serious or fatal re- 

 actions should horse serum or partially purified antitoxins be injected later. In fact, 

 the absence of serum reactions in many cases that had previously received toxin- 

 antitoxin indicates the safety of the procedure. 



A drawback to the use of the toxoid is that in children over seven years of age a 

 considerable percentage develop annoying reactions due to the proteins (meat extrac- 

 tives, peptone, and endocellular substances) in the toxoid broth. The o.i-L-f toxin- 

 antitoxin mixture contains only about one-fiftieth of the amount of these proteins. 

 However, the work of Watson and Wallace,' Moloney and Weld,^ and Watson and 

 Langstaff^ on the purification of toxoids is encouraging. These workers show that, 

 according to Ramon's fiocculation test, the antigenic substances can be obtained over 

 one hundred times purer than in the original toxin broth. If the active immunizing 

 value is retained in these products, the drawback of toxoid mentioned above can be 

 removed. Their method of purifying is essentially as follows: Diphtheria toxin de- 



' Watson, A. F., and Wallace, U.: /. Path. &° Bact., 27, 271. 1924. 



2 Moloney, P. J., and Weld, C. B.iloc. cit. 



3 Watson, A. F., and Langstaff, E.: J. Biochcm., 4, 50. 1926. 



