PRODUCTION AND TESTING OF ANTITOXINS 273 



the original quantity of serum. After filtration, this solution is 

 again precipitated with saturated ammonium sulphate solution and 

 the precipitate again filtered off. The precipitate is then treated 

 with a saturated solution of sodium chloride of double the volume 

 of the original serum. This is allowed to stand for about twelve 

 hours. At the end of this time the antitoxin-containing globulin 

 is in solution and is pipetted away from the precipitate and filtered. 

 This salt-solution extract is then precipitated with twenty-five 

 hundredths per cent acetic acid. The resulting precipitate of 

 globulin is thoroughly dried by pressure between filter papers and 

 placed in a parchment dialyzer. Dialysis with running water is 

 continued for seven to eight days, after neutralization with sodium 

 carbonate, in order to remove the sodium chloride. At the end of 

 this time, the globulin solution remaining in the dialyzer is filtered 

 through a Berkefeld candle for the purpose of sterilization, after 

 the addition of 0.8 per cent sodium chlorid. According to Gibson, 

 this method produces a yield of antitoxin which equals about four- 

 fifths of the original quantity but is concentrated five- to seven-fold. 

 The method has more recently been modified as follows : 



After heating to 56 C., as above, and cooling, ammonium sul- 

 phate is added to the serum to thirty per cent saturation. This 

 brings down all the euglobulins. This is then filtered and the filtrate, 

 which contains the pseudoglobulins with the antitoxin, is again pre- 

 cipitated with ammonium sulphate in a concentration of fifty-four 

 per cent of saturation. The precipitate is then separated on a paper, 

 pressed to dryness, and directly dialyzed. 12 



Park and Thorne 13 have found that the use of such concentrated 

 antitoxin is, therapeutically, equally efficient as the unconcentrated, 

 and possesses the advantage of less frequently giving rise to the 

 secondary reactions in skin and mucous membranes occasionally 

 noticed after the use of ordinary antitoxin, and referable, probably, 

 to some other constituent of the horse serum. 



Diphtheria antitoxin is therapeutically used in doses ranging 

 from 3,000 to 20,000 units. For prophylactic immunization of healthy 

 individuals, about 500 units should be used. 



See also the chapter on diphtheria for Schick reaction and active 

 immunization with toxin-antitoxin mixtures. 



12 Dr. Banzhaf, personal communication. 



13 Parfe and Thorne, Amer. Jour. Med. Sci., Nov., 1906. 



