Preparation of a polyclonal antibody against goldfish (Carassius auratus) vitellogenin and its application to detect the estrogenic effects of monocrotophos pesticide

Abstract

Goldfish (Carassius auratus) represents a good model to detect the estrogenic effects of chemicals, and vitellogenin (Vtg) is a vital indicator of estrogenic activity. The heterologous anti-carp Vtg antibody has previously been used for goldfish Vtg detection. Here, we report the preparation of an anti-goldfish Vtg antibody to improve the sensitivity and specificity of goldfish Vtg immunoassays. Vtg was purified from the plasma of 17β-estradiol (E2)-induced goldfish by gel filtration followed by anion-exchange chromatography. It was characterized as a phospholipoglycoprotein with an apparent molecular weight of ~460kDa and separated into three major polypeptides corresponding to ~130, ~106, and ~81kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). A polyclonal antibody against goldfish Vtg was raised in rabbits and found to be specific for goldfish Vtg through immunoelectrophoresis and Western blot. A sensitive sandwich enzyme-linked immunosorbent assay (ELISA) was developed for the quantification of plasma Vtg, with a detection limit of 3.6ng/mL and a detection range from 7.8 to 250ng/mL. The intra- and inter-assay coefficients of variations were 2.4–6.8% and 6.7–10.8%, respectively. Additionally, we qualitatively and quantitatively detected the induction of Vtg in male fish exposed to 0.01, 0.01, and 1.00mg/L monocrotophos pesticide by Western blot and ELISA. The homologous sandwich ELISA based on the anti-goldfish Vtg antibody could provide a valuable tool for the study of estrogenic effects of exogenous chemicals on goldfish.