PATHOLOGY: BERG AND KELSER 
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well known tetanus and diphtheria antitoxins are examples of preparations 
containing all or nearly all of the immunity units present in the original 
serums, but only a part of the proteins. Thus, Homer 1 (p. 400) concentrated 
a tetanus serum containing 100 units per cubic centimeter and 6% of protein, 
obtaining an antitoxin that contained 900 units per cubic centimeter and 19% 
of protein. In this process 10% of the antitoxic units were lost, the antitoxin 
was nine times as potent as the serum from which it was derived, but it con- 
tained only three times as much protein. The failure of all attempts to 
obtain a protein-free antitoxin preparation has led some investigators to the 
conclusion that the antibody or group of antibodies which constitutes the an- 
titoxin is one of the serum proteins and hence cannot be completely separated 
from protein. The concentration of the antitoxin without a similar concen- 
tration of protein is regarded by others as an indication that the antitoxin 
may be a body of non-protein nature. 
Under these conditions any test which would conclusively decide whether 
an antitoxin is or is not identical with a serum protein, would have both a 
practical and a theoretical interest. The following test was decided upon 
because of its promising nature. If an antitoxin, tetanus antitoxin, for ex- 
ample, is a substance of non-protein nature, it should be possible to prepare 
artificial digestion mixtures containing the antitoxic serum or antitoxin de- 
rived therefrom, in such fashion that the protein would undergo digestion 
without loss of antitoxin. Appropriate chemical measurements would indi- 
cate the extent of proteolysis, while inoculation experiments on guinea pigs 
would indicate whether there was any loss of antitoxic units. If, on the other 
hand, the antitoxin is a protein and its power to immunologically neutralize 
the corresponding toxin is a function of the intact protein molecule, then the 
antitoxin would be destroyed in every case where the protein had undergone 
cleavage, regardless of whether the cleavage was caused by a proteolytic enzyme 
or other chemical agent. Due regard must of course be had for the possible 
destruction of the toxin by the chemical agents used. 
Several different antitoxic preparations were exposed to the action of 
trypsin-sodium carbonate solution or to pepsin-hydrochloric acid for com- 
paratively long periods of time, with suitable controls. At proper intervals 
the extent to which digestion had taken place was measured chemically and 
compared with the loss of antitoxic units; the latter were determined by 
guinea pig inoculation as described by Rosenau and Anderson 2 . The results 
may be summarized as follows: 
1. Tetanus antitoxin in 0.5% sodium carbonate solution was slowly and 
completely destroyed. At the same time no significant chemical changes in 
the proteins were detected. 
2. In solutions amphoteric or faintly acid to litmus strips, trypsin destroys 
the antitoxin and at the same time the associated proteins are digested. The 
rates of antitoxin destruction and protein splitting were substantially the same. 
