METHODS IN MAMMALIAN IMMUNOGENETICS 377 



evolved rapidly into a variety of techniques suitable for the analysis of complex mixtures 

 of soluble antigens; for their identification and interrelationships and for the determina- 

 tion of the relative and absolute concentrations of the antigens, their diffusion co- 

 efficients, molecular weights, and electrophoretic mobilities. 



In 1956, Oudin immunized rabbits with immune precipitates (ovalbumin-rabbit- 

 anti-ovalbumin precipitate) plus Freund's adjuvants and demonstrated production of 

 isoantibodies which would precipitate serum components in some rabbits but not in 

 others, thus resulting in serum groups analogous to red blood cell groups. 977 The 

 term "allotypy" was proposed to designate the variation of the antigenic specificity of 

 these serum antigens. This work has opened a new approach to the genetic study of 

 proteins, one which we also have undertaken. 290 It has been demonstrated that these 

 allotypes may have electrophoretic mobilities of a-, (3-, and y-globulins; and that for 

 the y-globulins there are at least two genetic systems with at least three alleles at each 

 locus. In contrast to the polysaccharide antigens attached to red blood cells, which 

 have been studied by agglutination reactions combined with absorption methods as 

 cited by Dr. Owen, these soluble protein allotypes may also be studied by means of the 

 precipitin reaction utilizing the agar-gel immunochemical methods. Since the anti- 

 bodies are produced in the same species, specific antibodies are obtained without the 

 necessity of absorption techniques. It would not be unreasonable to expect that 

 other animal species would show the phenomenon of allotypy. Just as with the blood 

 groups based on differences of red blood cells, serum groups based on antigenic 

 differences of proteins should open new opportunities for work in mammalian genetics. 



I would like to make three additional comments on methodology in immuno- 

 genetics. The introduction of paraffin-oil adjuvants with Mycobacteria by Dr. Jules 

 Freund provided the most powerful immunization method known, at least for protein 

 antigens. 406 Such adjuvants have made it easier to produce antibodies to weak 

 antigens. One must keep in mind that this method of immunization introduces the 

 complication that very small amounts of antigen or contaminants will produce anti- 

 body whereas ordinarily they might not. Also, the antibodies produced with the use 

 of adjuvants may be more heterogeneous. 



When a mixture of several antigens are used for immunization, antibodies to each 

 antigen are not necessarily produced. In fact, competition may develop among the 

 antigens so that antibody production to one of the antigens, perhaps the one of interest, 

 may be suppressed. 2 Therefore, purified antigens should be used whenever possible. 

 Cellulose ion-exchange chromatography devised by Peterson and Sober is one of the 

 best methods now available for the fractionation of protein antigens. 999 



Finally, I would like to comment on the choice of species for antibody production. 

 Most commonly the rabbits or horses are used as recipients for preparation of anti- 

 bodies. However, a closely related species might be more revealing and perhaps more 

 sensitive to minor differences of antigenic properties. 289 This principle of immuno- 

 logic perspective was pointed out many years ago by Landsteiner. 749 



Dr. Klein : I would like to ask Dr. Winn whether the cytotoxic method is applicable 



