n8 THE PRINCIPLES OF IMMUNOLOGY 



the blood. The blood may be injected as defibrinated blood for purposes of im- 

 munization, but as a rule in order to avoid any influences the serum may have, 

 the blood is washed so that only corpuscles are injected. For purposes of washing 

 50 c.c. centrifuge tubes are desirable, but if these are not obtainable, the 15 c.c. 

 size may be employed. The blood is measured into the tube with a pipette, 

 usually to the amount of 5.0 c.c. The amount of blood is marked with a 

 grease pencil and the tube filled with physiologic salt solution. The tube is 

 centrifuged until the blood is thrown down. The supernatant fluid is poured off 

 and the tubes again filled with salt solution. The sedimented corpuscles are 

 shaken up into the salt solution and again centrifuged. This operation is re- 

 peated again, and the blood is said to have been washed three times. Aiter 

 the last centrifugation the supernatant fluid is poured off and the sedimented 

 blood-corpuscles restored to original volume by addition of salt solution. In 

 order to make a five per cent, suspension, this is washed into a 100 c.c. cylinder 

 and made up to 100 c.c. volume with salt solution. Any other percentage 

 desirable may be made by appropriate additions of salt solution to the original 

 blood mass. 



Preparation and Collection of Immune Sera. The injection into the rabbit 

 may be by subcutaneous, intraperitoneal, or intravenous routes. The intravenous 

 route produces immune bodies most rapidly, as has been shown by Bullock, 

 .and as a rule produces an immune serum of higher titer than is obtainable by 

 other methods. An excellent way to produce hemolysin rapidly is to inject in- 

 travenously into the rabbit three doses 4.0 c.c. each of 50 per cent, suspension 

 of washed sheep or goat erythrocytes at intervals of five days. The method of 

 intravenous injection has previously been described in connection with the pro- 

 duction of agglutinins (see page 82). A test bleeding may be made from the 

 posterior ear vein five to seven days after the last injection, and if the titer of 

 the serum is not sufficiently high, one or two more injections may be given. When 

 sufficiently high the rabbit is bled from the femoral artery as previously 

 described (see page 83). The blood is collected in a flask, the flask inclined 

 at an angle of about 45 until the blood is firmly clotted. The flask is then placed 

 in an upright position in the refrigerator for about twenty-four hours, after 

 which the collected serum is pipetted into a sterile container. Melick, in a study 

 of the influence of colloidal suspensions on the production of hemolysis, finds 

 that if he gives preliminary intravenous injection of aleurpnat suspension and 

 subsequently immunizes with blood-corpuscles, the hemolytic sera are of con- 

 siderably higher titer than in animals not so treated. 



Titration of Immune Sera. For titration of the hemolysin in the rabbit 

 serum, it is necessary to have a 5 per cent, suspension of the antigenic corpuscles, 

 the serum to be tested and irf addition fresh guinea-pig serum which serves as 

 complement. In such a titration the 5 per cent, suspension of corpuscles and the 

 complement are regarded as standards and employed in the same doses through- 

 out the series of tubes. If 0.5 c.c. of serum and 0.5 c.c. of corpuscle suspension 

 are employed, 0.05 c.c. of complement is usually sufficient, Before attempting 

 the titration the rabbit immune serum should be inactivated by heating in a 

 water bath at 56 C. for one-half hour. Dilutions of the inactivated immune 

 amboceptor are made as a rule i-io, i-ioo, 1-500, i-iooo, 1-1500, 1-2000, 1-2500, 

 1-3000, 1-4000. The guinea-pig serum (complement) is diluted i-io. The follow- 

 ing protocol will show how the series of tubes is set up : 



5 ^hee SP cells n Amboceptor Complement i-io Result 



0.5 c.c. -zoo 0.5 c.c. 0.5 c.c. CH 



0.5 c.c. -500 0.5 c.c. 0.5 c.c. 



0.5 c.c. -looo 0.5 c.c. 0.5 c.c. 



0.5 c.c. -1500 0.5 c.c. 0.5 c.c. 



O.5 C.C. -2OOO O.5 C.C. O.5 C.C. 



0.5 c.c. -2500 0.5 c.c. 0.5 c.c. CH 



0.5 c.c. -3000 0.5 c.c. 0.5 c.c. PH 



0.5 c.c. -4000 0.5 c.c. 0.5 c.c. 



0.5 c.c. 0.5 c.c. 0.5 c.c. 



0.5 c.c. i-ioo 0.5 c.c. 



The last two tubes are controls and should be made up to volume by addi- 

 tion of 0.5 c.c. saline in place of amboceptor in the one and of complement in the 

 other. The letters CH indicate complete hemolysis, PH partial hemolysis, and 



