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Journal of Agricultural Research 
Vol, VIII, No. 2 
demonstrated the value of anthrax serum as a curative agent and as a 
prophylactic when employed simultaneously with anthrax spore vaccine. 
Recalling the work on the separation of diphtheria antitoxin by frac¬ 
tioning the serum through the use of ammonium sulphate, the writers 
undertook the application of this method to anthrax serum and succeeded 
in producing the antibodies in a concentrated form. Chemical analyses 
of the serum and globulin preparations were made, and the changes in 
serum proteins during the course of hyperimmunization of animals 
against anthrax were studied. 
SEPARATION OF PSEUDOGLOBULIN FROM IMMUNE SERUM 
Themethod used was essentially similar to that described by Banzhaf (2) 
except that serum instead of citrated plasma was used. In all cases the 
serum was obtained from natural, spontaneously coagulated blood of two 
horses, which are here designated as horse 48 and horse 96. These had 
been hyperimmunized by the senior author, using an improved technic 
described in a previous publication (4). During the course of 6 months a 
total of 14 preparations of pseudoglobulin were made from 4 lots of serum 
from each horse. Serum 48 was known to have a high and serum 96 a 
comparatively low potency. 
In the beginning it was not known whether the antibodies in the anthrax 
serum would withstand heating to 6o° C., as in the diphtheria antitoxin 
preparation. For this reason each lot of serum was divided into two equal 
parts, one of which was given the heat treatment. Otherwise, the method 
of fractioning with ammonium sulphate, filtration, and dialysis was the 
same for both. The volume of serum used in each preparation varied 
from 600 to 1,200 c. c. (see Table I). Four preparations were simul¬ 
taneously made—two from serum 48 (heated and not heated) and two 
from serum 96 (heated and not heated). This was done in order that the 
results of the subsequent inoculation experiments might be comparable. 
The serums from.the two horses were not mixed, but were used separately 
in the pseudoglobulin preparation. 
The serum was diluted with one-half its volume of water, and saturated 
ammonium-sulphate solution was added up to 30 per cent saturation— 
that is, 30 per cent of the total volume of the mixture. Thus, to 1 liter 
of serum there were added 500 c. c. of water and 643 c. c. of saturated 
ammonium-sulphate solution. Thirty per cent of the total volume, 
2,143 c. c., consisted of saturated ammonium-sulphate solution, 643 c. c. 
At this concentration euglobulin was precipitated. 1 The mixtures to be 
heated were placed in an electrically heated drying oven maintained at 
6o° C. They were contained in 2-liter Erienmeyer flasks provided with 
rubber stoppers. With few exceptions they remained in the oven for 
six hours and were then filtered along with the corresponding mixtures 
1 In diphtheria antitoxin preparation the euglobulin carries down little, if any, of the antitoxin. 
Banzhaf ( 2 , p. 115). 
