430 THE DIPHTHERIA BACILLUS GROUP 
with antitoxin unimpaired, however. Toxin which is devoid of toxin 
power but which combines with antitoxin is called "toxoid." 
Antitoxin. — Prepam^^ow.— The injection of the soluble toxin, of the 
diphtheria bacillus in sub-lethal doses into experimental animals stimu- 
lates the formation of specific antitoxin which has both curative and 
prophylactic value. Antitoxin is usually obtained from horses. These 
animals are somewhat less susceptible to diphtheria toxin than many, 
but they are employed chiefly because a large yield of antitoxin is 
readily obtained from them. The serum of horses, at least in single 
doses, is innocuous for man, and horses furnish large amounts of blood 
(containing antitoxin) without injury to the animal. Young, animals 
free from glanders, tuberculosis and other diseases are used for the 
purpose. Several methods are available for immunization, but the one 
commonly selected is carried out in the following manner: an initial 
injection of diphtheria toxin, either mixed with an excess of antitoxin 
or attenuated by iodine trichloride, is made and about a week later a 
second injection containing an increased amount of toxin follows. At 
regular intervals the injections are repeated, each time increasing the 
amount of toxin in regular progression until after three to four months 
as much as 2,50 to 300 cc. of unaltered toxin is introduced at one time. 
After about two weeks following the last injection the animal is bled 
and the potency of the serum tested. If it contains 150 units or more 
of antitoxin to the cubic centimeter, from 2 to 5 liters of blood are 
removed from the jugular vein with sterile precautions into sterile 
receptacles, and the animal is again treated with toxin to induce further 
immunization. As a rule, about two-thirds of the volume of blood 
taken is regained in antitoxin-containing serum. It is customary in 
large establishments to immunize several horses at the same time and 
mix this serum, for experience has shown that the serum of certain 
animals contains substances which cause erythematous rashes in man 
which are disagreeable and irritating although not necessarily harmful. 
Pooling the blood reduces this possibility. The serum is stored in sterile 
containers in a dark cold place and retains its antitoxic properties well. 
It deteriorates less rapidly than toxin. 
Concentratio?i. — Atkinson^ noticed that the globulins of the horse 
serum increased and the albumins diminished as the antitoxin content 
of the blood increased, and he eft'ected a partial purification of the anti- 
toxin fraction by removing the albumin with protein precipitants. 
Recent work, however, indicates that the albumin fraction does not 
change materially although the globulin fraction increases parallel to 
the antitoxic increase.^ Gibson-^ carried the process further and ob- 
tained a serum which was about three times as rich in antitoxin per 
unit \-olume as the original horse serum. Banzhaf^ has reduced the 
1 Jour. Exp. Med., 1898, 3, 647. 
2 Kirkbride and Murdick: Jour. Immunol., 1927, 14, 235. 
3 Jour. Biol. Chem., 1906, vol. 1, Nos. 2 and 3. 
^ Collected studies from the Research Laboratory, New York City Board of Health, 
vols. 5 and 6, 
