Development and validation of an ELISA to measure vitellogenin in the zebrafish (Danio rerio)
Abstract
In this study, an enzyme-linked immunosorbent assay (ELISA) was developed to quantify vitellogenin (Vtg) in zebrafish (Danio rerio). Zebrafish Vtg (zf-Vtg) was purified from whole-body homogenates of estradiol-ex posed zebrafish, and polyclonal antibodies against zf-Vtg were raised. Using purified zf-Vtg as a standard and anti-zf-Vtg antibodies (DR-264), a competitive ELISA method was set up and validated, The working range of the assay is from 1 to 30 ng/ml (20-80% binding), and the detection limit is 0.4 ng/ml for purified zf-Vtg. In whole-body homogenates samples, the practical detection limit is higher than that for purified Vtg (40 ng/ml) due to matrix effect. The intra- and interassay variations were 4.7% and 14%, respectively, at 50% binding (n = 36). Its usefulness to detect changes in Vtg concentration in other cyprinid fish was also tested. In addition, the assay was used to assess Vtg induction in male zebrafish exposed to 17beta-estradiol (E-2). Exposure of male zebrafish to 0.1, 1, 10, and 100 mug/L of E-2 for 7 d led to a Vtg induction from the lowest concentration. The results show the suitability of the developed ELISA to quantify Vtg inductions in zebrafish, the cross-reactivity of DR264 antibodies with commonly used cyprinids, and the potential of zf-Vtg induction as a sensitive biochemical endpoint that could be used to detect estrogenic properties of chemical substances.