NATURE OF THE ACTIVE PRINCIPLES 303 



neutralizing antiI)odies is used, the precipitate as well as the 

 supernatant fluid contains the active principles. In filtrates+ 

 antibody mixtures, the precipitate formed may carry down the 

 non-neutralized actixe principles if present in amounts exceeding 

 the neutralizing potency of the serum. The process of mere ad- 

 sorption of the active principles by the precipitates, however, 

 leaves their potency unaltered. The neutralization of active prin- 

 ciples occurs in multiple proportions with a certain amount of 

 irregularity similar to that observed in diphtheria toxin-j-anti- 

 toxin neutralizations. It is possible however, to neutralize any 

 anioimt of active principles provided a sufficient concentration of 

 neutralizing antibodies is used. This is well substantiated by 

 neutralization exj^eriments with concentrated immune serum 

 (Chapter iv, p. 117). 



The protection against the active principles also can be ol)- 

 tained in vivo and protection thus obtained also occius roughly 

 in multiple proportions. The protection takes place in direct 

 multiple proportions until after a certain increase in the amoimt 

 of serum injected \vhen there occurs a two-fold and more than 

 t^vo-fold gain in the protective value of the serimi injected. The 

 gain may be due to a change in the rate of retention of the anti- 

 bodies in the general circulation following the increase in the 

 volume of serum injected. 



The facts considered together seem to be sufficient for pointing 

 out the close relationship of active principles to true bacterial 

 toxins as defined in the beginning of this chapter. The similarity, 

 however, also follows the physico-chemical properties of true 

 toxins, and the mode of their production. 



The chemistry of true toxins is only little known. In their 

 chemical behavior they have been long regarded as a protein- 

 like substance. The results of recent investigators tend to confirm 

 this view (Dernby, 1926; I.ocke and Main, 1928; and Eaton, 

 1936/;) . As stated by Eaton, all of the methods that can be suc- 

 cessfully used in the pmification of diphtheria toxin involve re- 

 actions more or less characteristic of proteins. The pinification of 

 true toxins in\'olves the separation of the protein fraction from 

 the chemically similar proteoses and peptones and other various 

 bacterial proteins. The methods used are precipitation by acids; 

 precijMtation at pH 5.0 with various negative ions; salting out 

 with ammonitnn sulphate; precipitation with alcohol and ace- 

 tone: purification by means of dialysis; adsorption; and finally 



