METHODS IN MAMMALIAN IMMUNOGENETICS 353 



purposes, sterile technique is not used in processing sera; the fresh serum itself is 

 effectively bactericidal, and the serum is generally stored frozen between periods of its 

 use. Preservatives are frequently added; Merthiolate (0.05 per cent) or sodium 

 azide (0.1 per cent) are most common. Mouse serum to be used for blood-typing tests 

 sometimes changes in its specificity or loses its activity upon ordinary storage; routinely, 

 mouse-typing reagents are therefore subdivided into small quantities in ampoules, and 

 are then lyophilized and stored in sealed ampoules, if possible after evacuation of the 

 ampoules. Most serologic typing reagents, however, are much less critical in their 

 storage requirements than are those prepared from murine serum. 



Immunization. — Numerous routes of injection are used; the amount and the 

 character of the antibody response is often influenced by the route adopted. Rabbits 

 are generally injected intravenously into the marginal ear vein. For immunization 

 with red-cell suspensions, we commonly inject 0.5 ml. of a 20 per cent suspension of 

 washed cells intravenously three times a week for three weeks. The rabbits are then 

 rested for 7-10 days before the antiserum is collected. The injection is made with a 

 f-inch, 25-gauge needle on a 2-ml. syringe; the use of inexpensive disposable syringes 

 and needles is becoming common and has several advantages. The amount injected, 

 the frequency of injection, and the duration of the immunizing period are arbitrary, 

 and other schedules are as effective as that described. Frequently, rabbits are kept 

 after their first bleeding, and after several weeks are injected again to produce more 

 antiserum. Very often the results of the later immunizations are better than those of 

 the first series. Because of the possibility of an anaphylactic response in the im- 

 munized rabbit when he is injected after an interval of rest, we usually make the first 

 reimmunizing injection intraperitoneally rather than intravenously, injecting about 

 four times as much material as is used in a single intravenous dose ; this has the effect of 

 desensitizing the rabbit, and two additional intravenous injections can then be made as 

 usual at two-day intervals. 



For some types of antigens, especially relatively small protein molecules of low 

 antigenicity, it has been found desirable to use adjuvants in connection with the 

 injections. Alum precipitation of the antigen before injection is often effective. 

 Another type of adjuvant very frequently used is based on the studies of Dr. Jules 

 Freund, and in its several modifications is generally referred to as Freund's adjuvant. 

 Procedures are described in connection with antiglobulin techniques in a later section 

 of this paper. 



Material in Freund's adjuvant is generally injected intramuscularly (in rabbits, 

 into the large muscle of a rear leg or into the loin), or subcutaneously (under the skin 

 of the back). Intense local reactivity is induced at the site of injection, and repulsive 

 necrotic lesions may appear. If the outer surface of the needle is kept free of Freund's 

 material, dry and sterile, such lesions are less frequent. An adjuvant often makes the 

 difference between getting a useful antiserum and getting none at all. Adjuvant, 

 however, in our hands generally has had little effect on circulating antibody responses 

 to red cells and similar large materials ; it would appear that such preparations generally 



