96 TOXINES AND ANTITOXINES. 



phate or of the hydroxides of the heavy metals could not be 

 used as means of isolating diphtheria antitoxine. Moreover, the 

 method employed by TISSONI for the isolation of his tetanus anti- 

 toxine (q.v.) viz., salting out with solid magnesium sulphate at 

 30 C. gave a yield of at most 50 per cent. On the other hand, 

 BRIEGER and BOER have obtained quantitative yields by the 

 following method : 



Ten c.c. of the immune serum are mixed with 10 c.c. of distilled water, 

 and the mixture treated with 4 grms. of dry potassium chloride, and 4 to 5 

 grms. of finely-powdered sodium chloride, and left for eighteen to twenty 

 hours in an incubating oven, after which the precipitate is dissolved in 

 water and dialysed. Frequently the precipitate agglomerates into nearly 

 a solid mass on stirring with water, and no antitoxine is dissolved. In 

 such cases it can only be extracted with weak alkalies. A more exact ex- 

 planation of this anomaly cannot be given. After the residue has been 

 dissolved, the solution is treated with an equal volume of finely-divided 

 magnesium sulphate, and again left for two to three hours in the incubating 

 oven, the result being that the antitoxine is quantitatively precipitated. 

 In this way they obtained 0*2 grm. of active dry substance from 10 c.c. of 

 the serum. 



BRIEGER and BOER have also endeavoured to concentrate the 

 antitoxine by precipitation with the salts of heavy metals. Zinc 

 salts proved to be the most suitable. 



The serum was diluted with five times its volume of water and treated 

 with twice the quantity of a 1 per cent, solution of zinc sulphate or zinc 

 chloride, and the precipitate washed with water. ^ It was then dissolved in 

 a ,^ N. solution of alkali hydroxide, and the zinc precipitated with 

 carbon dioxide. These experiments showed that in the precipitation with 

 zinc sulphate the antitoxine was precipitated with the zinc, while the zinc 

 chloride remained in solution. The portion containing the antitoxine was 

 dried in a desiccator, and it was found that the zinc albuminate was 

 somewhat soluble in water, while the zinc antitoxine was insoluble. The 

 latter was, therefore, again dissolved in alkali solution and the zinc once 

 more precipitated with carbonic acid. It was not possible to effect by 

 this means a complete separation of the antitoxine from the zinc. 



BRIEGER and EnRLicn 1 were the first to obtain active dry 

 preparations from the milk of immunised animals by means of 

 ammonium sulphate. They obtained one preparation which 

 contained 14 per cent, of ammonium sulphate and was 400 to 

 600 times as active as the original milk. 



WASSERMANN 2 has modified this method to some extent, 

 proceeding as follows : 



1 Brieger and Boer, " Beitr. z Kenntn. d. Milch immuner Tiere," Zeit. f. 

 Hyg., xiii., 336, 1893. 



a Wassermann, "Ueb. Concentrierung v. Antitoxin aus Milch, Zeit. f. 

 Hyg., xviii., 236, 1894, 



