766 TITRATION OF TOXINS AND ANTITOXINS 



sera and their flocculation value. The serum of one horse having the highest titre 

 in agglutinin for the streptococcus had the lowest antitoxic value as determined both 

 by flocculation and skin-testing methods. Other horses produced serum of high anti- 

 toxic quality with no demonstrable agglutinin for the streptococcus. Povitzky and 

 Dyer regard the flocculation reaction in this case as the result of the union in satu- 

 rating proportions of scarlatinal streptococcus toxin and its antitoxin. 



Flocculation titrations with the toxin and antitoxin of Streptococcus scarlatinae 

 have been found to be unsatisfactory by O'Brien, Okell, and Birkhaug (1926) and by 

 Eagles (1927). No constant relationship could be established by them between the 

 skin-test dose and flocculation titre. The bacterial precipitin reaction accounted for 

 the larger part of the precipitates formed. 



BOTULINUM TOXIN AND ANTITOXIN 



The evidence of the value of the flocculation method in the titration of botulinum 

 toxin and antitoxin is conflicting, Weinberg and Goy (1925) report that "le pheno- 

 mene de Ramon peut done servir egalement au titrage in vitro, du serum antibotulini- 

 que." In their tests they used 5 cc. of diluted toxin in each tube and varied the amount 

 of antitoxic serum. Typical flocculation occurred in three to four hours at 37° C. in 

 an indicating tube containing an approximately neutral mixture. Botulinum anti- 

 toxin, toxin, and a formalinized anatoxin were titrated in this manner. A potent toxin 

 was found to be essential, as the best results were obtained with toxins containing 

 200,000 to 600,000 M.L.D. per cubic centimeter. Antitoxic sera stored in the icebox 

 for two to four months had a greatly delayed and unsatisfactory flocculating power. 

 These investigators, as shown by their paper published in 1924, were well aware of 

 the precipitation which occurs when agglutinating sera are added to the filtrates of 

 bacterial cultures, but in the case of botulinum antitoxin and toxin they were able to 

 obtain satisfactory results by the flocculation method in spite of that difficulty. 



In the section dealing with the nature of the flocculation reaction, the failure of 

 the method to measure the values of botulinum toxin and antitoxin in the experiments 

 of Bronfenbrenner and Reichert (1926) has been referred to. The evidence presented 

 in this report clearly establishes the fact that an antibacterial serum, devoid of pro- 

 tective power, can cause a precipitate when mixed with a filtrate of an old culture of 

 CI. botulinum. The tests were made in such a manner, however, as would favor a pre- 

 ponderant bacterial precipitin reaction while masking specific toxin-antitoxin floccu- 

 lation. The unusually small quantities of toxin and serum used may have contributed 

 to the irregular results obtained. The toxin did not have as high potency as that em- 

 ployed by Weinberg and Goy and was strongly formalinized (0.7 per cent) and in- 

 cubated for a week before its use in the flocculating mixtures. This undoubtedly de- 

 layed flocculation. During the long periods of incubation without constant observa- 

 tion of the tubes, the initial flocculation may have been overlooked. Bronfenbrenner 

 and Reichert (1926) noted that the removal of the precipitate formed by the action of 

 a non-toxic botulinum filtrate upon an antitoxic serum did not materially reduce the 

 antitoxic value of the serum. This is an indication that the bacterial precipitin reac- 

 tion does not involve the antitoxin. Apparently no attempt was made to determine 

 whether such an absorbed serum would flocculate in a mixture with botulinum toxin. 



