MATERIALS AND METHODS 



Most blood samples were taken in the 

 field by severing the caudal artery with 

 a sharp knife and collecting the spurting 

 blood in sterile bottles. Some of the 

 samples, including those used in isoimmuni- 

 zations, were taken by cardiac puncture. 

 The clotted samples were maintained on ice 

 or in a refrigerator until used. Samples 

 excessively hemolysed or over ten days old 

 were not used for testing. Blood cells 

 from samples as old as three weeks were 

 used for animal inoculation. Cells for 

 testing were washed three times in 10 to 50 

 volumes of modified Alsever's solution 

 (Bukantz et al. 1946) and adjusted to a 2- 

 percent concentration in this solution. 

 The use of the Alsever's solution as a 

 suspending medium was found to be essential 

 since salmon red blood cells lysed in saline 

 or phosphate-buffered saline solutions. 



Most antisera were prepared by giving 

 intravenous or intraperitoneal injections 

 of 0.5 cc. of a 50 percent suspension of 

 washed cells. Such injections were given 

 to rabbits and salmon three times a week 

 for three weeks and to chickens every three 

 days for three or four injections. Four 

 to twelve days after the last injection the 

 cuiimal was bled and serum collected. In 

 most cases, in order to detect individual 

 differences, several additional stimulations 

 at intervals or two weeks to a month were 

 required. Some rabbits also received sub- 

 cutaneous inoculations of washed suspensions 

 of particulate material from lysed red 

 cells in Freund's adjuvcmt. The sera were 

 preserved by freezing and stored at -30° 

 C. Serum dilutions were made with phos- 

 phate-buffered saline or 1 percent saline 

 solutions. 



Absorptions were performed by mixing 

 a 1/2, 1/5 or 1/10 dilution of the heat- 

 inactivated antiserum with an equal volume 

 of washed packed cells, incubating for one 

 hour at room temperature or in the refri- 

 gerator, centrifuging the cells down and 

 decanting the absorbed serum. Usually more 

 than one absorption was required to remove 

 all of the antibody present which would 

 react with the absorbing cells. 



Tests were performed by mixing 0.1 ml, 

 of absorbed serum, diluted if necessary, 

 with 0.1 ml. of 2 percent cell suspension 



Table 1, — Individual antigenic differences demonstrated 

 by agglutinin absorption tests in the erythrocytes 

 of sockeye salmon from Cultus Lake and Adams River, 



(Rabbit antisockeye salmon-erythrocyte serum R19 absorbed 

 and cross-tested with the cells of individual salmon) 



Cultus Lake 1 

 Cultus Lake 15 

 Cultus Lake 19 

 Adams River 15 

 Adams River 11 

 Adams River 9 

 Adams River 13 

 Saline control 



in Alsever's solution in 10 x 75 mm. tubes. 

 Dilutions are expressed as the final dilu- 

 tion of the original serum, taking into 

 account the dilution by the red cell sus- 

 pensions. Readings were made after suit- 

 able incubation periods at room temperature 

 and usually after overnight incubation in 

 the cold. The settling pattern was judged 

 either smooth (S) or rough (R) and the 

 degree of agglutination scored as 0, +_, 1 

 plus, 2 plus, 3 plus and 4 plus. In order 

 for a test to be considered, the saline 

 control had to be smooth and negative. 



RESULTS 



The major part of our effort has been 

 directed toward finding blood types in red 

 or sockeye salmon ( Oncorhynchus nerka 

 Walbaum). Toward this end we have immunized 

 26 rabbits and 20 chickens with the washed 

 erythrocytes or stroma from members of this 

 species. For each of the other species of 

 Pacific salmon we have made 2-8 rabbit and 

 chicken anti-erythrocyte sera. Individuals 

 or pools from individuals from the same 

 area were used. These antisera were ab- 

 sorbed with the erythrocytes of individual 

 salmon from other areas and the resulting 

 sera tested for residual activity for the 

 cells of a number of individuals. With most 

 antisera produced, numerous absorptions did 

 not reveal any evidence for antigenic het- 

 erogeneity within this species. However, 

 blood group differences were demonstrated 

 with a few rabbit immune sera. One of these 

 sera (R19) was analyzed by absorption with 

 the cells of sockeye salmon collected in 

 1955 from Cultus Lake and the Adams River, 



