(Cumley and Irwin, 1943) and have not been 

 used in racial studies. It would therefore seem 

 that antigenic differences in serum proteins 

 offer little promise for racial analysis in fish. 

 Nevertheless, the proper application of the new 

 serological techniques known as agar diffusion 

 may demonstrate useful intraspecific differences. 

 Serum proteins have one advantage over red 

 cells in that they are easily preserved by freez- 

 ing and can be held for much longer periods of time. 



From the work done on other animals and 

 man, it seems that the immunological techniques 

 most likely to yield results which can be used 

 in racial analyses in fish are those used to 

 demonstrate intraspecific differences in cellular 

 antigens. These are illustrated in figure 1 and 

 consist of giving experimental animals a course 

 of injections with washed blood cells from in- 

 dividual fish of the species being investigated. 

 The surface of these red cells contains high- 

 molecular -weight substances which are foreign 

 to the experimental animal and consequently 

 serve as antigens, i.e., they cause the animal 

 to respond by producing specific antibodies 

 against them. This process is identical to vac- 

 cination. As we all know, vaccination against 

 typhoid does not make us immune to diphtheria; 

 this is because the antibodies produced are so 

 specific that they will react only with the antigen 

 which induced their production or with very 

 closely related antigens. In the illustration, 

 the different surface antigens are characterized 

 diagrammatically as different geometric shapes, 

 some of which vary between the cells of the two 

 individual fish and some of which are present on 

 the cells of all members of the species . The 

 specificity of the antibodies formed is illustrated 

 by indentations in them so that they fit their 

 corresponding antigens much as a hand fits a 

 glove or a key fits a lock. In order to make the 

 antisera thus produced useful in differentiating 

 between fish of the two types, we must remove 

 all the antibodies to the antigens which are com- 

 mon to the two types. This is accomplished by 

 the technique of absorption. The antisera to 

 type FA is mixed with red cells of type FB and 

 correspondingly the antisera to type FB is mixed 

 with red cells of type FA. When this process is 

 properly carried out, all the antibodies to com- 

 mon antigens are removed when the red cells 

 are centrifuged down. Only type specific anti- 



bodies remain in the serum - that is, they will 

 agglutinate only red cells which possess the 

 antigen which characterizes one antigenic type. 

 These antibodies are not removed because they 

 do not fit any of the antigens present on the red 

 cells used for absorption. Sera which are pre- 

 pared in this manner can be used to determine 

 the antigenic types of individuals in populations 

 of interest and to determine the frequency of 

 occurrence of these types. If statistically sig- 

 nificant differences in these frequencies are 

 found, we have tools for studying the geographic 

 range, extent of straying, precision of homing 

 and other characteristics of the populations in 

 question . Of course, the greater the number of 

 antigenic characteristics which we have sera to 

 test for, the more likely we are to find differ- 

 ences in frequencies between populations of 

 interest. 



The illustration shows rabbits as the ex- 

 perimental animal used for antibody production . 

 However, one should use other animals as well, 

 preferably animals as closely related to the 

 donor as possible . The ideal procedure, and 

 one which has been used to demonstrate many 

 individual antigenic differences in man, cattle, 

 sheep, chicKens and goldfish, is isoimmuniza- 

 tion. This consists of using members of the 

 same species for both donor and recipient. 

 Antigens possessed by the donor and not the re- 

 cipient are foreign to the latter and antibodies 

 will be produced to these substances just as they 

 would be to any other foreign antigen . Finer 

 antigenic differences are usually demonstrable 

 by isoimmunization and, of course, antibodies 

 against species -specific antigens are not formed 

 so they need not be removed by absorption . 

 When using fish as an antibody former, one must 

 keep in mind the fact that their rate of antibody 

 production is dependent on the temperature of 

 the water in which they are held (Snieszko et al., 

 1938) (Cushing, 1942). 



One of the biggest technical problems dur- 

 ing developmental work to discover red cell 

 antigens is the necessity for having fresh red 

 cells for testing the antisera and for absorptions. 

 In the case of fish, especially pelagic and anadro- 

 mous fish, it is not always possible to obtain 

 blood samples repeatedly from the same individual. 

 Two methods of preserving red cells which were 



40 



