762 TITRATION OF TOXINS AND ANTITOXINS 



and 6, and by adding w/i NaOH to increase the pH to 8 and 9. When the toxic broth 

 was made more acid or alkaline than it was at the time of original filtration, non- 

 specific precipitates were produced in the broth, which obscured any flocculation due 

 to the union of toxin and antitoxin. Removal of the precipitate by filtration or cen- 

 trifugation reduced the Lj value of the toxin. The results of titrations, therefore, at 

 different pH values beyond the range 6.4-8.4 were irregular and of no significance. 



Chemical agents. — Toxoids produced by the action of chemicals flocculate like 

 those produced by age and heat up to 40° C, retaining substantially the same Lj 

 value which the original toxin possessed. Their time of flocculation, however, is pro- 

 longed. An excessive amount of such substances as iodine and formaldehyde, which 

 deprive toxins of their poisonous properties while leaving their antigenic qualities 

 intact, may inhibit flocculation. Formaldehyde, in excess of five parts of commercial 

 40 per cent formalin to 1,000 cc. of toxin, may prevent flocculation. In this case, the 

 formaldehyde combines with amino acids and other constituents of the toxic broth as 

 well as with the toxin. The properties of the non-specific combinations cannot be pre- 

 dicted. It is, therefore, advisable to control the addition of formaldehyde by the 

 flocculation reaction, as Ramon does in the preparation of the non-toxic immunizing 

 agent which he calls "anatoxin." 



Both toxins and antitoxic sera have properties, not understood at present, 

 which influence the rate of flocculation. Usually toxins of high potency and fresh 

 sera flocculate most rapidly. But the sera of some horses flocculate only slowly 

 in spite of a high antitoxic value. Several specimens of antitoxic serum, plasma, and 

 pseudoglobulin may all contain the same number of antitoxic units per cubic centi- 

 meter and yet differ by many hours in the time in which they flocculate with the same 

 toxin at the same temperature. These differences may be due to protective colloidal 

 properties of the fluids, or to differences in antigenic potency of the toxins and a qual- 

 ity of the serum known as its "avidity." The rate of flocculation of different toxins, 

 toxoids, and anatoxins, when tested against the same serum, has been shown by 

 Ramon (1922-26), Weinberg and Goy (1924), Descombey (1925), and Glenny, Pope, 

 and Waddington (1925) to give an indication of the immunizing value of the prepara- 

 tion. The best antigens are those which flocculate most rapidly and have a high Lf. 

 Madsen and Schmidt (1926) have recently published experiments relating the speed 

 of flocculation of antitoxins to their neutralizing speed and curative efficiency. This, 

 therefore, connects the speed of flocculation with the "avidity" of an antitoxin, as 

 sera which flocculated more rapidly were curative in lower doses as measured by anti- 

 toxic units than more slowly flocculating sera. 



RELATIONSHIP OF FLOCCULATION VALUE TO OTHER UNITS 



The flocculation reaction is theoretically a measure of the combining value of all 

 the substances in a toxic broth capable of uniting with antitoxin. Most of the com- 

 parisons between the various units established by animal tests and those obtained by 

 flocculation have been made with diphtheria toxin and antitoxin, and from them the 

 following generalization may be drawn : 



There is no relationship between the actual toxicity of a toxin or of any unit based 

 upon that toxicity and its flocculating value, except possibly in the filtrates of young 



