Observations were also made upon the 

 reactions of fresh tuna erythrocytes with human 

 anti-A and anti-B typing serums. Table 7 shows 

 that the little tunny and oceanic skipjack show 

 very little reactivity with these serums, while 

 the reactions of the yellowfin are much more 

 marked. However, as both anti-A and anti-B 

 serums react almost equally well, normal anti- 

 bodies must be involved that are not specifically 

 related to the human blood types. Absorption of 

 anti-A serum with yellowfin cells, while re- 

 moving all agglutinins for these cells, was not 

 observed to reduce the titer of the anti-A serum 

 with respect to human A cells. The prelimin- 

 ary observations on stroma noted above and the 

 recollection of the relationships among the 

 human A subtypes shows that this is not a con- 

 clusive observation. Further observations upon 

 fresh yellowfin cells could not be made, so that 

 the nature of the A-like substances on tuna cells 

 still remains to be elucidated (Cf . Gushing and 

 Sprague, 1953, for an earlier discussion of this 

 problem). A final observation in this connection 

 is that the antiserums in table 7 that were 

 titrated (/) were not able to agglutinate human A 

 cells in spite of their reactivity for the cells of 

 tuna. 



Tuna stroma . Much attention was given 

 to the preparation and agglutination of washed 

 tuna stroma in the hope that these might be use- 

 ful in studies where fresh red cells could not be 

 obtained. However, it was found difficult to ob- 

 tain consistent preparations and to achieve 

 agglutinations to any usable degree. As a re- 

 sult of many observations the conclusion was 

 reached that future efforts should be made to 

 work with intact erythrocytes rather than to 

 develop techniques using frozen whole bloods in 

 which the cells had been hemolized. 



Preservation of intact erythrocytes . 

 The above conclusion diverted research from 

 frozen tuna blood to efforts to preserve intact 

 erythrocytes by freezing in glycerol. This 

 method has been successfully applied to the 

 preservation of human erythrocytes by Chaplin 

 and MoUison (1953) in England. While fresh 

 tuna bloods were not available, it was possible 

 (with Mrs. Elyse Beaver) to show that small 

 aliquots (appx. 1 to 3 ml.) of the cells of shiner 



seaperch ( Cymatogaster aggregata Gibbons) 

 could be preserved by a modification of this 

 technique, and that the cells of other species 

 also gave promising results. At present it may 

 be concluded that the glycerol -freezing tech- 

 nique should be investigated whenever it becomes 

 desirable to make and preserve large scale col- 

 lections of tuna blood from diverse areas . 



Forssman antigen. Three anti-tuna serums 

 were tested for their ability to hemolize sheep 

 cells in the presence of guinea pig complement. 

 The hemolytic titers observed after 15 minutes 

 at 37°G. were as follows: anti -yellowfin 1, 

 1:1280; anti -oceanic skipjack, 1:640; anti -pacif- 

 ic mackerel, 1:5120. 



Tuna antigens occurring in white croakers 

 and shiner seaperch . Cells of the shiner sea- 

 perch ( Cymatogaster aggregata Gibbons) were 

 found to be agglutinated by anti -yellowfin tuna 

 serum 2 to a titer of 1:4096. Conversely, cells 

 of the white croaker ( Genyonemus lineatus Ayres) 

 were found to be agglutinated to a titer of 1:400 

 by the anti-oceanic skipjack serum already re 

 ferred to in this paper. Shiner seaperch cells 

 were also weakly agglutinated by this second 

 serum, but this agglutinin could be absorbed, 

 leaving the white croaker agglutinin intact. 

 Table 11 shows the results of absorbing a mix- 

 ture of anti -yellowfin 2 and anti -skipjack serum 

 with the washed stroma of individual yellowfin 

 and skipjack . The preliminary absorption of 

 skipjacK antiserum with shiner seaperch cells 

 was necessary to remove the low -titer agglutin- 

 ins for these cells . The results show that 

 yellowfin tuna and skipjack are actually distin- 

 guishable by two antigens found on white croaker 

 and shiner seaperch cells respectively. 



As table 7 shows, variations exist among 

 the species of tuna examined with respect to re- 

 actions with anti-albacore and anti -yellowfin 

 tuna serums; therefore, the two antigens under 

 discussion would seem to be of potential inter- 

 est in evolutionary investigations along the lines 

 discussed in Gushing and Sprague, 1953 . The 

 possibility also exists that antigens of such wide 

 distribution among diverse species may be of 

 value in searching for individual antigen varia- 

 tions within single species of fish. For example. 



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