PRODUCTION OF ANTISERUM 



The antigenic substance used for production of 

 antisera was a vitellin preparation from starry floun- 

 der eggs. This prei)aratiou met the chissical bio- 

 chemical criterion for vitelhn, since it was the water- 

 insoluble fraction of the egg yolk (Jukes and Kay, 

 1932). The pr('])aration was made in tlie following 

 manner: extraction was in a Waring Blendor ^ from 

 1 part of eggs to 3 parts 1 percent saline solution. 

 One part of the supernatant oi)taincd after centri- 

 fugation at 10,000 r.p.m. for 20 minutes was diluted 

 with 11 parts distilled water, and the resulting pi'c- 

 cipitate was dissolved in 1 percent saline, rcprecip- 

 itated, and redissolved. The vitellin preparation 

 was utilized in this final form for immunization 

 procedures. Altiiough different injection routes were 

 used and the vitellin preparation was injected both 

 with and without iidjuvants, consistently uniform 

 antisera were produced in the five rabbits that were 

 stimulated. Because of this uniformity, a single 

 pooled reagent composed of nvmierous bleedings 

 from all of the rabbits was made and used through- 

 out this study. 



The antibody specificities appeared to be directed 

 toward one or more of the starry flounder's vitellin 

 antigens. The pooled antiserum cross-reacted with 

 serum of mature females of all pleuronectid species 

 tested. Reactions with sera from males were ob- 

 served only infrequently. These reactions were 

 invariably weak and are discussed in a later .section. 



COLLECTION OF SAMPLES 



Collection dates and numbers of English sole 

 sampled are listed in table 1. All samples were 

 collected by the University of Washington research 

 vessel Commando at the Port Orchard area of Paget 

 Sound. Only female English sole were taken. 

 These fish, with the exception of the March sample, 

 were randomly sampled over a variety of lengths. 

 This sample was biased toward smaller individuals 

 with relatively undeveloped ovaries. This bias was 

 based on the relatively high frequency of the HM 

 factor in smaller females in the two previously col- 

 lected samples; the data for the March sample reflect 

 this bias. Random sampling was resumed through 

 the remainder of the investigation. 



Collection of an adequate blood sample from h^ng- 

 lish sole was found impractical, and all (lualitative 

 determinations for the HM factor were made from 



the fluids expressed from kidney tissue. When 

 quantitative data were required, blood samples were 

 collected from larger individuals by cardiac i)uncture. 

 The collection data for the halibut samples are 

 presented in table 2. A sufficient blood volume was 

 available from the individual halilnit to u.se serum 

 for determination of the HM factor, although par- 

 allel samples of kidney tissue were colle('ted from 

 most fish. All samples were collected under the 

 direction of the International Pacific Halibut 

 Commission. 



'l'.\BI,?; 1. — Collection (Uilix (111(1 number of Joiidh EiKjlish sole 

 in samples, Port Orchard area, Wash. 



Table 2. — Date and areas of collection, number of indindu(d 

 samples, and source of HM antigen for halibut 



* Trade names referred to in this publication do not iniitly cndorscnient 

 of commercial i>roducts. 



' Serum collected day of capture; fiah eviscerated and iced at tliis time 

 and kidney fragments collected in port 4 days later. 



AGE DETERMINATIONS 



Age determinations in both halibut and English 

 sole were made from otoliths. Personnel of the 

 International Pacific Halibut Commission made age 

 determinations of the halibut. English sole otoliths 

 were treated in papain as described by Pruter and 

 Alverson (l'J()2) and read by the senior author. 



COMPARISONS OF HM CONCENTRATION 

 IN SERUM AND KIDNEY FLUIDS 



Tlie need to u.se kidney tissue to obtain ciualitative 

 data on the HM factor in English sole required us to 

 compare concentrations of the HM factor in kidney- 

 tissue fluids and blood serum of the same individuals. 



50 



U.S. FISH AND WII.DLIFK SEUVICK 



