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Assessment of in situ estrogenic effects in the Black chinned Tilapia (Sarotherodon melanotheron) using vitellogenin measurement by ELISA method
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The aim of this study was to develop an enzyme linked immunosorbent assay (ELISA) to quantify vitellogenin in the Black chinned Tilapia (Sarotherodon melanotheron). Tilapia vitellogenin T-vtg) was first purified from plasma samples of estradiol-induced fish using a two-step chromatographic method. The T-vtg was characterized with an estimated molecular weight of 482 kDa under native conditions. Polyclonal antibodies against T-vtg were raised in rabbits and the specificity of the antibodies to recognized T-vtg was verified by Western blotting and indirect ELISA experiments on blood samples. By using these antibodies and purified T-vtg, a competitive ELISA was set-up and validated. This assay appears sensitive, with a detection limit of 1.81 ng/ml, and specific, as shown by the competition curves, obtained by serially diluting blood of females, that were parallel to the T-vtg standard curves. The ability of the T-vtg ELISA to quantify vtg induction was achieved by in situ experiment. For this purpose, adults males and females tilapia were collected in 8 West African sites and circulating vtg concentration was measured. The present study shows that the developed T-vtg ELISA is a useful tool to assess the effect of estrogenic contamination in wild Black chinned Tilapia.
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