352 



FISHERY BULLETIN OF THE FISH AND WILDLIFE SERVICE 



temperature and 30 seconds centrifngation. In 

 the preparation of specific, agglutinating reagents, 

 antisera were absorbed by incubating one part 

 redfish cells and four parts diluted antiserum for 

 10 minutes at room temperature. One absorption 

 was usually sufficient to remove all antibodies 

 reactive with the absorbing cells. Eeagents and 

 antisera were frozen in 3-ml. aliquots which were 

 thawed and heat- inactivated just before use to 

 destroy complement. 



RESULTS 



Rabbit antisera prepared by injecting pooled 

 and individual samples of redfish cells provided 

 the first indication of individual differences in 

 redfish erythrocytes. At appropriate dilutions of 

 these antisera, cells of certain fish reacted weakly, 

 while cells of other fish were strongly positive. 

 As an example, a rabbit antiserum (labeled 

 GBR17E), prepared by injecting pooled cells of 

 14 Georges Bank redfish, agglutinated cells of 

 certain redfish from a small sample taken at 

 Eastport, Maine, to a titer of 1 : 128, while cells 

 from other fish in the same sample were not ag- 

 glutinated beyond the 1:32 dilution (table 1). 

 The degree of agglutination was recorded conven- 

 tionally in descending order from (+ + + +), 

 representing complete agglutination, to ( — ), 

 indicating no agglutination. 



Table 1. — Rcuctioiu of rabbit anti-rcdfifih scrum 

 (GBR17R) with erythrocytes of si.r individual Eastport 

 redfish 



Table 2. — Results of absorbing^ rabbit anti-red fish scrum 

 (GBR17R) tcith erythrocytes of six individual Eastport 

 redfish 



Cells from fish Nos. 1 and 4 reacted weakly 

 with the antiserum, while cells of other fish re- 

 acted more strongly. This suggestion of individ- 

 ual differences in redfish erytlarocyte antigens was 

 .supported by absorptions of tlie antiserum. Re- 

 sults of absorbing with cells from each of the fish 

 mentioned above are presented in table 2. 



.Vbsorption of the antiserum with cells of fish 

 Nos. 2, 3, 5, and fi removed antibodies for absorb- 

 ing cells and for all others, while absorption with 



I .\bsorptions of specific antibodies were made by incubating one part 

 redfish cells and 4 parts diluted (1:4) antiserum lor 10 minutes at room tem- 

 perature. After this period, the absorbing ceUs were centrifuged and the 

 supernatant fluid tested against an aliquot of the cells used for absorption. 

 One absorption was usually sufficient to remove all antibodies reactive with 

 antigens of the absorbing cells. 



cells of fish Nos. 1 and 4, which reacted weakly 

 with unabsorbed antiserum, removed antibodies 

 for each other, but left antibodies which aggluti- 

 nated cells of fish Nos. 2, 3, 5, and 6. Comparison 

 of cell agglutinations with absorbed and miab- 

 sorlied antiserum indicated that absorptions drasti- 

 cally reduced the antibody content. The tests 

 demonstrated antigenic dissimilarity in redfish 

 erythrocytes, but suggested that the antigens were 

 closely related. As a descriptive measure, fish 

 whose cells reacted strongly with unabsorbed anti- 

 serum, and also removed antibodies for all other 

 cells in absorptions, were considered as possessing 

 an antigen Ai, while those fish whose cells reacted 

 weakly with the miabsorbed antisertmi, and did 

 not remove antibodies for Ai cells in absorptions, 

 were considered as possessing an antigen A;. 



Ref eriiiig again to table 2, weakly reacting cells 

 of fish Nos. 1 and 4 would thus be A,, while 

 strongly reacting cells of fish Nos. 2, 3, 5, and 6 

 would be Ai. Using a reagent prepared by ab- 

 sorbing the GBR17R antiserum with cells of Ao 

 fish, it was possible to recognize fish of each anti- 

 genic type, since only cells of fish with Aj antigen 

 were agglutinated. 



Confirmation of this system was possible with a 

 rabbit antiserum (labeled MIC8R) which had been 

 prepared for another study by injecting pooled 

 cod cells. Individual differences were sliglit with 

 the unabsorbed antiserum, but absorption with 

 cells of the six fish used previously produced re- 

 sults shown in table 3. 



Absorption with cells of fish Nos. 1 and 4 re- 

 moved antibodies for absorbing cells and for all 



