EDWIN J. BANZHAF 757 



euglobulin-like substance as it was formed. This method showed a further conversion 

 of about 10 per cent. During 191 2 the writer added 0.5 per cent phenol to the anti- 

 toxin plasma before adding the ammonium sulphate. The addition of phenol short- 

 ened the necessary heating period and aided greatly in the subsequent filtrations. 

 Briefly the method is as follows: 



The antitoxin plasma containing 0.5 per cent phenol is diluted with a half-volume of 

 water and 30 per cent of a saturated ammonium sulphate solution is added. The mixture 

 is heated at 57° C. for one hour and then rapidly brought up to 63° C. It is cooled to about 

 50° C. and filtered. The precipitate contains the euglobulin. The filtrate called "A" is 

 measured and brought up to a 45 per cent content of saturated ammonium sulphate solution. 

 This will precipitate all the antitoxin and pseudoglobulin. After standing about three hours, 

 the mixture is filtered through hardened filter papers or muslin cloths. The first precipitate 

 containing the euglobulin (see above) is added to a volume of water equal to half the original 

 volume of plasma used. This is precipitated with 32 per cent saturated ammonium sulphate 

 solution. In this "washing" of the euglobulin it is necessary to use a slightly higher percent- 

 age of saturated ammonium sulphate solution as the protein content is decreased, which will 

 increase the solubility of the euglobulin.^ After filtering, the washed euglobulin precipitate 

 is discarded. To this second filtrate, called "B," saturated ammonium sulphate solution is 

 added up to 45 per cent. The resulting precipitate is collected by filtration through the 

 hardened filter papers containing the main precipitate from filtrate "A." After filtration is 

 completed, the combined precipitates are pressed to remove as much ammonium sulphate 

 solution as possible and dialyzed to free it from this salt. In the dialysis the accumulated 

 water dissolves the precipitate. One per cent sodium chloride and 0.4 per cent trikresol are 

 added. (The required amount of trikresol is added to a like amount of ether. The mixture 

 can be added to protein solutions without causing any appreciable coagulation.)* The 

 dialyzed antitoxin product is stored in a cold room for about three to four months, to allow 

 for the separation of lipoidal substances which are usually present in the solution. After 

 storing it is clarified by filtering through paper pulp and filtered through a Berkefeld candle 

 to remove bacteria. Practically all the dialyzed antitoxin products concentrated by this 

 method have lipoidal substances in solution. After some months these substances become 

 insoluble and aggregate into a mass which rises to the surface, forming a cream-colored ring 

 on top of the antitoxin solution. The writer's investigations (unpublished work) show that 

 these lipoidal substances are extracted from the euglobulin fraction in the serum by the 

 action of the ammonium sulphate and heat, and are soluble with the pseudoglobulin which 

 is soluble in 30-33 per cent saturated ammonium sulphate solution. When the saturated 

 ammonium sulphate solution is added in sufficient amount to precipitate the antitoxin, these 

 substances are also precipitated. Ammonium sulphate in sufficient amount to precipitate the 

 greater part of the euglobulin fraction has also, but to a lesser degree, a solvent action on the 

 lipoidal substances without the aid of heat. The lipoidal substances may be completely 

 avoided in the antitoxin solution by the following method of concentration: 



Antitoxin in plasma containing 0.5 per cent phenol is diluted with an equal volume of 

 water and heated as previously stated. Cooled to below 37° C, another volume of water is 

 added equal to the amount of original plasma and then saturated with sodium chloride. This 

 will precipitate all the euglobulin and render the lipoidal substances insoluble. Allow to 

 stand four or more hours after complete saturation and filter. To this filtrate "A" is added 

 35 per cent saturated ammonium sulphate solution. This amount of sulphate in the presence 



' Gibson, R. B., and Banzhaf, E. J.: loc. cit. 



^ Krumwiede, C, and Banzhaf, E. J.: /. Infect. Dis., 28, 367. 192 1. 



