250 IMMUNOLOGY 



servation of Renaux (1924) that a nonflocculating antitoxin can 

 be titrated by the flocculation test if it is mixed with a standard 

 flocculating serum. The results obtained indicate the combined 

 neutralizing value of the mixture and from this the strength of 

 the unknown can be calculated. Neither Bayne- Jones (1928) nor 

 Ramon noted the Danysz phenomenon in any of their experiments. 

 The former reports (1928) that the optimum temperature for con- 

 ducting the test is 45°-55° C. The speed of flocculation increases 

 with the temperature up to 55° C. Above this temperature the 

 results are irregular. At 0° C. to 5° C. the speed of precipitation 

 is sufficiently slow to permit setting up the te$t and allowing the 

 tubes to remain in the refrigerator overnight without a flocculent 

 precipitate forming. Definite results are then obtained by incubat- 

 ing at 45° to 50° C. 



Eaton (1936) presents an interesting report on the flocculation 

 reaction with purified diphtheria toxin. He offers evidences that 

 the flocculation rate of purified toxins is unclianged when tlie 

 toxins have not been altered by the methods used in purification. 



pH of Toxin. — The pH of the toxin can vary, according to 

 Bayne-Jones, between 6.8 and 8.4 without affecting the results 

 of the flocculation test. Toxoid, as well as toxin, readily produces 

 flocculation of antitoxin. The Lf dose of toxin represents its 

 antigenic or combining power rather than its toxic strength. In 

 the case of a fresh toxin containing practically no toxoid the Lf 

 dose will equal the Lo dose of toxin. As the Lo dase of toxin is 

 converted gradually to toxoid, the toxicity for guinea pigs dimin- 

 ishes but its flocculation value remains the same. This is true 

 even when it is completely converted to toxoid. 



Reliability of R.vmon Test, — Since the Ehrlich method of 

 titrating toxin determined the toxic rather than the antigenic 

 strength of a toxin, it is evident why the Ramon flocculation rather 

 than the Ehrlich method is used to measure the strength of toxoid 

 which is nontoxic but antigenic. The test is, however, not always 

 a satisfactory index of the antitoxic property of a serum, since 

 the sera of some horses flocculate slowly and yet possess a high 

 antitoxic content. On the other hand, a rapidly flocculating 

 standard antitoxic serum is considered quite reliable in determin- 

 ing the flocculating dose of toxoid. 



