DIPHTHERIA 217 



in full bottles in the refrigerator. Before use it is tested on guinea- 

 pigs. If the material contains an adequate amount of toxin, less 

 than 0.01 c.c. should kill an animal, weighing about 250 grams. 



Since the injection of the crude toxin often gives rise to quite 

 severe local as well as systemic reactions, various attempts have 

 been made so to modify the material as to diminish this feature as 

 far as possible without interfering with its antigenic value. This 

 has been accomplished, in a measure, by giving the horse a large 

 dose of antitoxin mixed with the first three doses of toxin. In the 

 laboratories of the Health Department of New York City the 

 animals thus receive as initial dose an amount of toxin sufficient 

 to kill 5000 guinea-pigs (average weight 250 grams), i. e., about 

 20 c.c., and mixed with this 10,000 units of antitoxin. This injection 

 (given subcutaneously) is followed by a febrile reaction which lasts 

 for three to five days, when a second injection of a slightly larger 

 dose is given, and after a similar period of time a third one, both 

 being accompanied by a dose of 10,000 antitoxin units, as in the first 

 instance. After that the immunization is continued with increasing 

 doses of toxin, given by itself, and at intervals of five to eight days, 

 until at the end of two months from ten to twenty-times the original 

 amount is given (Park). If during this period the animal should 

 at any time react unduly by fever, or if any infiltration should occur, 

 it is recommended to resume the combined administration of toxin 

 with antitoxin, as in the beginning. At the expiration of six weeks 

 or two months the animal's blood is tested for its content in anti- 

 toxin. If by that time this has reached a titer of 100 to 150 units 

 the animal may be expected to ultimately furnish a serum of moder- 

 ate strength. If high-grade sera only are desired, it is needless to 

 continue with any animal that at this period does not give a titer 

 which is higher than 150. 



After this test bleeding, immunization is further continued with 

 increasing doses, at intervals of three days to a week, until the 

 animal furnishes a serum with the titer that is desired, or until this 

 can no longer be increased. At the end of three months two or 

 three animals out of fifteen or twenty will give a titer of about 500 

 units, and half of the total number one of 180 to 200. Further 

 injections may increase the production still farther, but it is note- 

 worthy that values of 500 to 600 units are rare. Higher values than 

 1000 are very uncommon, and Park states that of his horses not a 



