Jan. 8,1917 
Immunity Studies on Anthrax Serum 
39 
that had not been heated. Hard papers were used (S. & S. 575, 24 cm. 
diameter) in glass funnels. The filtration was fairly rapid, not trouble¬ 
some, and was allowed to go on overnight. The precipitated euglobulin 
was rejected. The filtrate contained the anthrax-immune bodies, 
pseudoglobulin and albumin. 
The ammonium-sulphate content of the filtrates was now raised to 50 
per cent saturation—that is, sufficient was added so that one-half of the 
final volume of the mixture consisted of saturated ammonium-sulphate 
solution. Thus, 1 liter of euglobulin filtrate contained 300 c. c. of satu¬ 
rated ammonium-sulphate solution, 233 c. c. of water, and 466 c. c. of 
serum. The addition of 400 c. c. of the saturated solution resulted in 
1,400 c. c. of a mixture containing 700 c. c. of saturated ammonium- 
sulphate solutions, 233 c. c. of water, and 466 c. c. of serum. Such a 
mixture is “50 per cent” saturated. At this concentration pseudo¬ 
globulin was precipitated, carrying with it the, anthrax-immune bodies. 
The mixtures were filtered as before, on hard papers. The filtrates 
containing albumin were rejected. 
The precipitated pseudoglobulin was then freed from most of the 
adherent liquid by pressure between filter papers and towels, the pre¬ 
cipitate being retained in the filter paper opened to form a semicircle. 
A convenient arrangement was the following: A towel; on top of this a 
large ordinary filter paper or two; on this, two filter papers containing 
precipitates. These two were covered with two ordinary filter papers 
and a towel, and so on. The pressure at first must be very slight and 
the filter papers and towels frequently renewed. After two or three days 
of pressure with gradually increased weights the precipitates were trans¬ 
ferred to a press which pressed them into a condition resembling soft 
cheese. The color was white or nearly white. The pressed precipitates 
were easily removed from the papers with a spatula, were weighed, and 
transferred to parchment dialyzing bags. The weights varied, of course, 
with the quantity of serum used and other factors. From a liter of 
serum about 125 gm. of moist, pressed pseudoglobulin were obtained. 
From 1 to 3 c. c. of chloroform were added, depending upon the amount 
of precipitate, and the bag was tied. The pseudoglobulin was dialyzed 
for 3 to 4 days against running tap water until only small amounts of 
sulphate were present in the tap water. The under surface of the bags 
was submersed in the tap water to a depth of 1 or 2 cm. 
At the end of the dialyzing period the bags were opened and the volume 
of the globulin concentrate was measured. In all cases the reaction to 
litmus-paper strips was either neutral or faintly amphoteric; the odor of 
chloroform had disappeared, showing that the chloroform had dialyzed 
out and that there is no great danger of using too much chloroform in the 
beginning. The concentrates were odorless or nearly so. They were 
transferred to glass bottles and kept in a refrigerator. As a preservative 
0.5 per cent chloroform was added. This has been found to be a most 
