THERAPEUTIC IMMUNIZATION IN MAN 459 



can be made directly in these syringes or in test tubes, and are 

 allowed to stand one hour at room temperature, so that there 

 may be time for complete union. If the mixtures are made di- 

 rectly in the syringes the needles are dipped into sterile vaselin, 

 which closes them and prevents, leakage while standing. The 

 mixture is then forced out of the syringe with a rubber bulb, thus 

 ensuring complete injection of all the fluid. As Rosenau states, 

 much depends on the guinea pigs. They must be of standard 

 weight, about 250 grammes, well fed and cared for, and must not 

 be descendants of pigs that have shown marked or unusual resist- 

 ance to diphtheria toxin. This, as Theobald Smith has shown, 

 occasionally happens. 



The antitoxic serum as obtained from the horse directly may 

 be concentrated in a number of ways, representative of which is the 

 method developed at the New York Department of Health by Gib- 

 son, 23 Banzhaff, and others. 24 The original method consisted in 

 heating horse serum to 56 C. for 12 hours, by which some of the 

 pseudoglobulin was converted into euglobulin, the antitoxin remain- 

 ing in the pseudoglobulin fraction. After this an equal volume of 

 saturated ammonium sulphate solution is added and the globulin 

 precipitated. After several hours the precipitate is filtered off and 

 again taken up in water corresponding in amount to the original 

 volume of serum. After filtration this solution is precipitated with 

 ammonium sulphate and this precipitate is treated with saturated 

 solution of NaCI in quantity twice that of the original serum. After 

 standing for 12 hours the supernatant fluid containing the antitoxin 

 is decanted, and this is precipitated with 0.25 per cent, acetic acid. 

 The resulting precipitate is dried by pressing it between filter papers 

 and is placed in a parchment dialyzing bag, after neutralization with 

 sodium carbonate. At the end of seven or more days of dialyzation 

 .against running water, the globulin solution remaining in the dial- 

 yzer is filtered and made isotonic. 



More recently the method as modified by Banzhaff is as follows : 

 The serum, as obtained from the horse, is diluted by one-half the 

 volume of water, and to this a saturated solution of ammonium 

 sulphate is added up to 30 per cent, saturation. This is heated to 

 61 C. for two hours. It is then filtered and the residue on the filter 

 paper, which contains the antitoxin, is thoroughly dried by pressing 

 hetween filter papers and is directly dialyzed. 



Observations by Park and Throne 25 have shown that this con- 

 centrated antitoxin which, according to Gibson, represents a yield of 

 about YO per cent, original antitoxic power of the serum, is equally 

 efficient for therapeutic purposes as an unconcentrated preparation 



23 Gibson. Journ. of Biol Chem., Vol. 1, 1906. 



24 Gibson and Collins. Journ. of Biol Chem., Vol. 3, 1907. 



25 Park and Throne. Amer. Journ. of Medical Science, Vol. 132, 1906. 



