| Environmental estrogens have directly threaten on the existing of wildlife and health of human beings. The study on the mechanisms and screening methods of which has been the hot topics in environmental research. Recently, vitellogenin(Vtg) has been studied extensively as a biomarker for the screening of environmental endocrine disrupting chemicals.Vitellogenin production can provide a valuable biomarker of reproductive disruption in fish. Abnormally high or low levels are indicative of disruption at multiple sites of the reproductive endocrine system, or of early sexual development and differentiation. Indeed, induction of Vtg is now the most widely used indicator for estrogenic exposure in fish.The zebrafish (Danio rerio), a member of the family Cyprinidae and native to India and Pakistan, has, during the last decade, been intensively used as an experimental animal for biological and biotechnological research. In addition, the zebrafish is a species routinely employed in the Organization of Economic Cooperation and Development(OECD) toxicity tests.The objective of the present study was to develop a homologous, indirect competitive enzyme-linked immunosorbent assay (ELISA) for Vtg and validate its use for the detection of estrogenic responses in zebrafish. In order to overcome the difficulties of obtaining sufficient Vtg from such a small fish, we use of Lv as antigen instead of Vtg for antibody production.Another problem normally interfering between small sized fish and the use of the ELISA for Vtg, is the minute amount of plasma that can be withdrawn from the fish. Thus,it is often difficult to achieve enough plasma for repeated or even single ELISA measurements. To solve this problem, the ELISA for Vtg can be based on the use of the supernatant of whole body homogenate (WBH). In this case, one must take into account, that the anti-zebrafish Lv IgG will recognise both Vtg and Lv. Therefore, it is necessary to use male fish if the endpoint is Vtg.The yolk protein, lipovitellin (Lv) was purified from ovaries of mature female zebrafish( Danio rerio) by Sephacryl S-300.Polyclonal antibodies against Lv were raised in rabbits.A gel electrophoresis by SDS-PAGE followed by Western blot analysis on Lv and samples of WBH from untreated males and females with removed gonads showed clear affinity of the anti-Lv IgG only to Lv and females.This indicates that the antibody wasspecific to only Lv and Vtg. This indicates the use of Lv as antigen and standards as a reliable substitute for Vtg in this assay.Anti-Lv IgG was used to develop a indirect competitive enzyme-linked immunosorbent assay (ELISA) to measure Vtg concentrations of whole body homogenate (WBH) in zebrafish.Adult male zebrafish were exposed to a nominal water concentration of 17b-estradiol (2.5, 5,10,20,80 andl60ng/l), 4-nonylphenol (10,25,50 and 100μg/L) and bisphenol A (50,100,200,500,1000,2000 and 4000μg IV) in a semi-static exposure system for 14 days. The estrogenic effects of treatment were investigated on vitellogenin by ELISA. Compared with the control group, all these compounds were found to be estrogenic to zebrafish.To study the effects of mixed exposure of nonylphenol (NP) and bisphenol A (BPA) on vitellogenin induction in zebrafish. Adult male zebrafish were exposed to different concentration mixture of NP and BPA :62.5μg/LNP+500μg/LBPA,62.5μg/LNP+1000μg/LBPA, 62.5μg/LNP+2000μg/LBPA, 125μg/LNP+500μg/LBP A, 125μg/LNP+1000μg/LBPA, 125 μg/LNP+2000μg/LBPA.The results showed that the combined effect of NP and BPA was greater than the sum of their individual effects. The interaction of NP and BPA was synergism action.
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