CHAPTER LVI 



THE TITRATION OF TOXINS AND ANTITOXINS 

 BY THE FLOCCULATION METHOD 



STANHOPE BAYNE-JONES 



University of Rochester 



If a physico-chemical titration method coul4 be substituted for, or used largely 

 to supplant, the testing of toxins and antitoxins upon animals, great economies in time 

 and materials would be made, and the precision and character of the procedure would 

 yield new information concerning the nature of these substances. The flocculation 

 method, described in its present form by Ramon in 1922, seemed to offer these ad- 

 vantages. Since the publication of his paper, the precipitation of bacterial toxins and 

 antitoxins in appropriate mLxtures has been used for the practical titration of toxins 

 and antitoxins, for the estimation of the immunizing values of toxoids and anatoxins, 

 for the preparation of a non-toxic antigen, for the purification of toxin and antitoxin, 

 and for the investigation of their properties and quantitative relationships. The evi- 

 dence accumulated from these numerous studies substantiates the opinion that a 

 specific flocculation occurs with regularity when a bacterial toxin is mixed in suitable 

 proportions with its antitoxin and that the reaction serves to measure the binding or 

 total antigenic value of the toxic filtrate as well as the neutralizing capacity of the 

 antitoxin. Recent criticisms of the method have again focused attention upon the 

 mixture of bacterial proteins and other residues in these filtrates which react to form 

 precipitates with the antibacterial substances present in the sera obtained from ani- 

 mals immunized by injections of them. While this type of reaction may occasionally 

 introduce confusing zones of precipitation, its occurrence does not exclude the specific 

 mutual flocculation of toxins and antitoxins in mixtures in the region of their neu- 

 trality. 



Flocculation in approximately neutral mixtures of ricin and antiricin was observed 

 first by Danysz in 1902. The precipitate in these "optimum" mixtures contained all 

 the ricin and antiricin o]:iginally present in the fluids. In 1909 Calmette and Massol 

 measured the protective value of antivenoms by a flocculation method and showed 

 that the precipitate contained both cobra venom and its antivenom in a dissociable 

 combination. Their work was followed by the experiments of Nicolle, Cesari, and 

 Debains (1920), in which results conformable with animal tests were obtained by 

 observing the precipitation which occurred at the junction between a layer of anti- 

 toxic serum upon a toxin incorporated in gelatin. A simplification of these procedures 

 was made by Ramon in 1922 when he found that an abundant flocculent precipitate 

 occurred in mixtures of diphtheria toxin and antitoxin in equivalent proportions. To 

 a series of tubes containing a constant amount of toxin he added decreasing amounts 

 of antitoxic sera. After one or more hours at room temperature, an opalescence and 

 tuie precipitate occurred in several of the tubes. This increased slowly, and at the end 



759 



