NOTE Hiroishi et al.: Identification of Pagus ma/or eggs using monoclonal antibodies 



557 



perature for 15 minutes. Then 

 the stick was washed with 0.1% 

 Tween 20/PBS, and incubated 

 with 4-chloro-l-naphthol solution 

 (substrate of horseradish peroxi- 

 dase in the kit) containing 0.1% 

 H 2 2 at room temperature for 

 15 minutes. The immunoglobu- 

 lin subclass of the monoclonal 

 antibodies was determined by 

 observing the positions of bands 

 that appeared on the stick. 



Results and discussion 



After cell fusion, hybridomas 

 were grown in 42 wells of 96- 

 well plates. Supernatant solu- 

 tions of the cultures were used 

 for the immunostaining assay 

 to select hybridomas producing 

 antibodies reactive to P. major 

 eggs. After the assay, positive 

 reactions were observed in six 

 wells. These hybridomas were 

 cloned by the limiting dilution 

 method, and finally three clones 

 producing monoclonal antibod- 

 ies reactive with P. major were 

 obtained. Those antibodies were 

 named MT-1, MT-2, and MT-3. 

 The subclass of all antibodies 

 was IgGj. Specificity of the anti- 

 bodies was examined by using 

 the eggs shown in Table 2. As a 

 result, the antibodies were reac- 

 tive with all the P. major eggs 

 in both the early and late stages 

 (before or after tail-bud stage), 

 but not with eggs of other species 

 (Table 3, Fig. 1). Thus, it becomes 

 possible to identify P. major eggs. 

 The immunostaining assay took 

 2.5 hours. 



The oldest eggs of P. major (20 

 April, 1995) could react with the 

 antibodies obtained as clearly 

 as the recently collected eggs 

 of P. major, indicating that egg 

 samples preserved for up to 7 

 years could be analyzed by this 

 method. 



The method was also success- 

 ful with 102 eggs collected from 

 Wakasa Bay (Table 4), which 

 had been immediately fixed with 

 5% formaldehyde in seawater. 

 Among them, only 11 eggs were 

 identified as Callionymoidei spp. 



