22 
The proper quantit}^ of the toxine and an amount of the antitoxin to 
be tested are now mixed in a test tube, as follows: To 0.05 c. c. of the 
toxine is added 0.001 c. c. of the serum, and the mixture inoculated 
suhcutaneousl}" into a guinea pig. If there was sufficient antitoxin in 
the 0.001 c. c. of the serum used to neutralize the 10 MLD’s used, the 
pig would survive, and it was then considered that 0.001 c. c. of that 
serum contained at least one-tenth of an immunity unit, and that 1 c. c. 
of that serum would consequently contain at least 100 units. 
Attention is particular!}^ called to the fact that at first the strength 
of antitoxin depended upon the complete neutralization of the poison 
as determined by indications of sickness in the guinea pig, particularly 
changes at the site of inoculation. Slight swellings which disappear 
in a few days were not taken into consideration in determining the 
value of the serum. It is evident that the indications of sickness in 
the guinea pig or the presence of slight changes at the site of inocula- 
tion are dependent upon subjective considerations leading to difi'er- 
ences of opinion or errors in judgment which could materially ,afl‘ect 
the accuracy of the tests. 
A partial neutralization of the poison sufficient to save the life of 
the animal is a more definite factor than the complete neutralization 
depending upon the appearance of local signs for recognition. 
On account of these difficulties encountered in determining the value 
of antitoxins it became necessary to eliminate all subjective considera- 
tions and obtain a method based upon strictly objective conditions. 
Both Ehrlich and von Behring independently came to the conclusion 
that the death of the animal was a much better criterion for determin- 
ing the value of a serum than the local reactions or the survival of the 
animal heretofore depended upon. 
A still more radical change in the principle of measuring the 
strength of diphtheria antitoxin was now introduced. 
Up to this point the basis of measuring the strength of diphtheria 
antitoxin was always either the culture of the diphtheria bacillus or 
its poisonous products. The genius of Ehrlich disclosed errors in the 
methods heretofore described. His researches into the biological 
relations between toxine and antitoxin showed that it required vary- 
ing amounts of antitoxin in order to neutralize the 10 MLD’s. He 
finally showed that there is no relation between the poisonous efi'ects 
of the toxine and its power of combining chemically to neutralize the 
antitoxin. He showed that a toxine may be weakened very materially 
by the influence of time, light, heat, oxygen, and other deleterious 
influences, without, however, altering its power of combining with 
antitoxin. This discovery was of fundamental importance and re- 
quired the transfer of the standard of measurement from the poison 
to its antibody. 
