STANHOPE BAYNE- JONES 767 



These results demonstrate that botulinum toxin and antitoxin present special difficul- 

 ties from the point of view of fiocculation titration, but do not conclusively controvert 

 the evidence on the side of the specificity and usefulness of this reaction. 



TETANUS TOXIN AND ANTITOXIN 



Although Descombey (1924, 1925) demonstrated that fiocculation occurs in a 

 typical manner in mixtures of tetanus toxin and antitoxin, and that formalinized ana- 

 toxins (toxoids) having a high flocculating value are more efficacious as immunizing 

 agents than similar preparations with a low flocculating value, other investigators 

 have found the method somewhat unsatisfactory. Scholz (1924) found that the small- 

 est differences measurable by the fiocculation titration of tetanus antitoxin were three 

 to four units, whereas he was able to detect differences of 0.0 1 unit by animal tests. 

 Abt and Erber (1926) obtained good results with 90 per cent of the preparations tested 

 by them. Of thirty-eight sera, 60 per cent of the antitoxins gave exactly equal values 

 by fiocculation and animal tests; in 29 per cent, the flocculating values were somewhat 

 higher than the results of the animal tests, and in 11 per cent the values indicated 

 by flocculation were lower than those of the tests on mice. Their opinion was that in 

 view of the irregularity of the results of animal tests when a small number of animals 

 are used the flocculation results were more precise than those of the animal tests. 

 Zone phenomena, the freshness and potency of toxins and antitoxins, the effect of 

 chemicals, heat, and age upon these reagents, and all the factors which influence floccu- 

 lation with diphtheria toxin and antitoxin come into play in this case also. In their 

 tests, Abt and Erber placed in each tube 4 cc. of tetanus toxin containing 20,000 

 M.L.D. per cubic centimeter, as determined on white mice, and added varying 

 amounts of antitoxin. At 45° C. in the water bath, the optimum temperature, floccu- 

 lation usually occurred in two to three hours. Ramon and Descombey (1926) have 

 proved that the mixture in the indicating tube in the flocculating series is a neutral 

 one when injected into mice. They discovered that not all antitoxic sera are satis- 

 factory for this test, but with the serum from one horse, obtained at monthly intervals 

 during the past three years, their flocculation titrations have had the desirable degree 

 of precision and have aided them in the choice of the most efficacious toxins and ana- 

 toxins for the immunization of horses. With the proper choice of toxin and antitoxin, 

 this type of titration can be depended upon to yield satisfactory results. 



DYSENTERY TOXIN AND ANTITOXIN 



Flocculation in an "indicating" tube has been observed by Dumas, Ramon, and 

 Said Bilal (1926) in mixtures of filtrates of cultures of the Shiga type of dysentery 

 bacillus and the antitoxic sera of horses immunized by injections of this toxin. The 

 mixture in the indicating tube was non-toxic, showing that neutralization had oc- 

 curred, but in the absence of well-established units of toxicity or protective values, it 

 is difficult to compute the exact significance of the proportions involved. The experi- 

 ments afforded additional evidence of the specificity of the toxin-antitoxin fioccula- 

 tion reaction, as the antitoxic serum did not contain demonstrable agglutinins for the 

 Shiga bacillus or any other type of dysentery bacillus. Filtrates were deprived of their 

 toxicity by the addition of 0.6 per cent of commercial 40 per cent formaldehyde, being 



