72 METHODS FOR EFFECTING ACTIVE IMMUNIZATION OF ANIMALS 



injections should be filtered to remove small particles of fibrin, and pref- 

 erably washed four times with sterile salt solution. The method of 

 preparing corpuscles for injection has been given in a preceding chapter. 

 After the third or fourth injection the animal should be bled from the 

 ear and the serum tested, as animals not infrequently succumb after 

 the fourth injection, and many possess serums of high potency after re- 

 ceiving three injections. By this method success is better assured. 



Intravenous Method. For the preparation of most hemolysins 

 the following methods yield very good results. Antihuman hemolysin 

 is more difficult to prepare, and I have generally found that a slower, 

 intraperitoneal method will yield better results. 



First Method. Four injections of 5 c.c. each of a 10 per cent, sus- 

 pension of washed cells at intervals of three days. The animal is bled 

 three or four days after receiving the last injection. 



Second Method. Three injections of 10 c.c. each of a 10 per cent, 

 suspension of washed cells on each of three successive days. The rab- 

 bit is bled four days after receiving the last injection (after the method of 

 Gay). Instead of these large injections, 1 c.c. of corpuscles, removed 

 after thorough centrifugalization and diluted with sufficient sterile 

 salt solution, may be given on each of three successive days. 



Third Method. A slower method consists in making the injections of 

 a suspension of corpuscles every five days. The cells must be thoroughly 

 washed to free them from all traces of serum; if this is not done, the 

 animals may die of anaphylaxis during the course of immunization; 

 animals should be tested after the third dose has been injected: 



First dose: 3 c.c. of a 10 per cent, suspension of corpuscles. 

 Second dose: 5 c.c. of a 10 per cent, suspension of corpuscles. 

 Third dose: 10 c.c. of a 10 per cent, suspension of corpuscles. 

 Fourth dose: 15 c.c. of a 10 per cent, suspension of corpuscles. 

 Fifth dose: 20 c.c. of a 10 per cent, suspension of corpuscles. 



Fourth Method. In the preparation of antihuman amboceptor, 

 Noguchi advises giving four intravenous injections of washed cells, 4 c.c., 

 3 c.c., 4 c.c., 3 c.c., and possibly another 4 c.c. at intervals of four or 

 five days; these amounts of packed cells are suspended in 10 c.c. of sterile 

 salt solution. Rabbits are bled one week after the last injection is given. 



Intraperitoneal Method. In this method the most careful aseptic 

 precautions should be observed in washing the cells and in giving the 

 injections, or the animals are likely to succumb from peritonitis just 

 about the time they are fully immunized and ready for bleeding. In- 

 jections may be given every four or five days, and one week after re- 

 ceiving the last injection the rabbits are to be bled. 



