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Fishery Bulletin 102(3) 



isolation tanks at several sea farming centers described 

 in Table 2. Immediately after collection, fish eggs were 

 fixed in a solution of 57c formaldehyde to sea water solu- 

 tion and stored. Before use, the eggs were thoroughly 

 washed with distilled water and suspended in phosphate 

 buffered saline (PBS) solution. 



Monoclonal antibodies were developed according to 

 the methods of Kdhler and Milstein (1975), Garfre and 

 Milstein (1981), and Hiroishi et al. (1984, 1988): 0.5 mL 

 of egg suspension (200 eggs/PBS solution from Fukui 

 Prefectural Sea Farming Center, Obama City, Fukui 

 Prefecture) was mixed with 0.5 mL Freund's complete 

 adjuvant (Nacalai Tesque, Inc., Kyoto, Japan). The mix- 

 ture were then injected subcutaneously into BALB/c 

 female mice (4 weeks of age). The female mice received 

 second and third injections at 2-week intervals. For the 

 final immunization, P. major eggs collected in the sea 

 farming center of Kansai Environmental Engineering 

 Center Co. (Miyazu City, Kyoto, Japan) were injected 

 into the mouse after being emulsified with Freund's 

 incomplete adjuvant (Nacalai Tesque, Inc.). Three days 

 after the final immunization, the spleens of the mice 

 were removed and passed through a mesh (mesh size: 

 100 urn). The spleen cells obtained by this procedure 

 were fused with the myeloma cell line X63-AG8.653 

 at a ratio of 10:1 with 50% polyethylene glycol. After 

 cell fusion, hybrid cells were incubated in a selective 

 hypoxanthine-aminopterin-thymidine medium (Kohler, 

 1979; Garfre and Milstein, 1981). 



The reactivity of the antibodies produced by the hy- 

 bridomas was then determined. Eggs fixed with 57c 

 formaldehyde in seawater were washed with PBS solu- 

 tion in a 96-well plate. Throughout the experiments, 

 the principal eggs used were from the Fukui Prefec- 

 tural Sea Farming Center. Normal horse serum solution 

 (200 jUL), diluted 100-fold with PBS, was added to the 

 wells to prevent any nonspecific reactions. After incuba- 

 tion at room temperature for 20 minutes, the eggs were 

 washed with 200 pL of PBS. After removing the PBS, 

 200 fih of the hybridoma culture supernatant solution 

 was added to the wells and incubated at room tempera- 

 ture for 30 minutes. After washing with PBS (100 /jL), 

 biotinylated horse anti-mouse IgG (100 pL) was added 



to the wells and incubated at room temperature for 20 

 minutes. After the incubation, VECTASTAIN R ABC re- 

 agent (avidin DH + biotinylated horseradish peroxidase/ 

 PBS, 100 jjL) was added according to the direction of 

 VECTASTAIN R Elite ABC kit (ABC Mouse IgG Kit, Fu- 

 nakoshi Co., Tokyo, Japan). After immunostaining the 

 eggs were observed by stereoscopic microscopy ( SMZ-2T, 

 Nikon Co., Tokyo, Japan). In a positive reaction, the 

 surface of the fish egg was stained brown as a result 

 of the oxidation of 3,3'-diaminobenzidine (substrate) 

 by horseradish peroxidase bound to the egg surface by 

 the antibody. 



Unidentified pelagic fish eggs from open water were 

 collected by using a plankton net (MTD net, NGG54 

 with mesh size of 0.344 mm, Rigo Co., Tokyo, Japan) 

 from Wakasa Bay (Fukui Prefecture, Japan) in May 

 1997. They were fixed with 59c formaldehyde in sea 

 water, either immediately or after incubation in seawa- 

 ter in finger bowls at 20°C for 24 hours, and identified 

 by careful observation as described by Ikeda and Mito 

 (1988) and Ikeda et al. (1991). The fixed eggs were 

 transferred to net wells (mesh size 200 ^m, diameter 24 

 mm, Corning Incorporated, Corning, NY) and washed 

 with 10 mL of distilled water three times. Then the 

 eggs in the netwells were immersed in 100 mL of PBS 

 in a polystyrene tray (Corning Incorporated, Corning, 

 NY) for 5 minutes. The egg suspension was placed into 

 the wells of a six-well plate and incubated with 10 mL 

 of normal horse serum solution for 20 minutes. After 

 incubation, the eggs were incubated with 10 mL of MT-1 

 antibody solution (hybridoma culture supernatant) and 

 then incubated with 10 mL of biotinylated horse anti- 

 mouse IgG. The subsequent procedure was performed 

 as described above. 



The immunoglobulin subclass of monoclonal antibod- 

 ies was determined according to the directions of the 

 mouse monoclonal antibody isotyping kit (Amersham 

 Pharmacia Biotech Co., Uppsala, Sweden) as follows: 

 3 mL of monoclonal antibodies solution (hybridoma 

 supernatant solution) obtained in this study was added 

 to 0.3 mL of horseradish peroxidase-conjugated anti- 

 mouse IgG in the kit. An isotyping stick in the kit 

 was incubated with the above solution at room tern- 



