METHODS IN MAMMALIAN IMMUNOGENETICS 363 



we do not ordinarily purify it more than one further precipitation and resolution, and 

 we recognize that we have more than y-globulin in this preparation. We then alum- 

 precipitate the protein for injection according to the following procedure. 



Dissolve the globulin prepared as above from 40 ml. of rabbit serum, in 25 ml. 

 buffered saline. Add 80 ml. distilled water, then 90 ml. of 10 per cent potash alum in 

 water. Adjust with NaOH to pH 6.5. Let stand ten minutes, centrifuge and wash 

 once with saline. Resuspend in 15 ml. buffered saline, and divide into three equal 

 aliquots. Inject one (intramuscularly) ; freeze the other two aliquots to be injected 

 (i.m.) respectively one and two weeks later. Bleed after an additional two-weeks' rest. 

 The goat can be restimulated by injecting globulin in solution, or even by injecting 

 whole rabbit serum. 



Alternatively, the globulin or whole serum can be injected in Freund's adjuvant. 

 The procedure we use is essentially that described by Munoz. 919 To 2 parts of paraffin 

 oil (Drakeol 6-VR, obtained from the Pennsylvania Refining Company, Butler, 

 Pennsylvania), containing 2 mg. of heat-killed and lyophilized M. tuberculosis (we use an 

 avirulent strain H37Ra, obtained from Dr. Sidney Raffel of Stanford University), is 

 added one part of Falba (obtained from Pfaltz and Bauer, Incorporated, Empire State 

 Building, New York 1, New York). After autoclaving, this mixture is taken at 3 parts 

 of the sterile adjuvant to 2 parts of the salt-precipitated globulin. If this is, for ex- 

 ample, mouse globulin to be injected into a rabbit, we prepare three ampoules, each 

 with 1 ml. of the adjuvant-globulin mixture, and inject the contents of one ampoule 

 subcutaneously once each week for three weeks, then bleed after a rest of two weeks. 



There are other methods for making antiglobulin sera and conducting anti- 

 globulin tests, some of them better for particular systems than others. References, 

 and a quick and clear survey of the field, can be found in Race and Sanger's book. 1036 

 Coombs and Roberts 226 have published a brief review, including further applications 

 and adaptations of the method. 



5. Elimination rates of labeled cells. A useful serologic technique is based on the 

 accelerated clearance of labeled antigen from the circulation of immune animals. 

 With soluble antigens, this is one of the more sensitive and convenient indicators of the 

 immune state. 205, 278 The method has found little application in immunogenetics, 

 although it offers promise. Some use has been made, however, of the clearance of 

 labeled red cells, and the observations at hand should stimulate further study. The 

 label of choice appears to be Cr 51 . Mollison, 888 who has contributed actively to this 

 area, has reviewed the methodology recently and has called attention to rather frequent 

 evidence of incompatibility based on reduced survival of Cr 51 -labeled human cells 

 injected into normal human recipients in whose serum no incompatible antibodies 

 could be found. An example of fruitful application of a similar technique to 

 problems of immunogenetic incompatibilities in mice is a paper by Goodman and 

 Smith. 453 



6. Tests on mixtures of cells. As mentioned earlier, the saline-agglutination 

 system, hemolysis, and the antiglobulin system (in the case of rhesus red-cell mixtures) 



