Development of enzyme-linked immunosorbent assays for plasma vitellogenin in Chinese rare minnow (Gobiocypris rarus)

Due to the wide occurrence of endocrine disrupting chemicals in the environment, it is much of importance to develop high throughput screening method for the analysis of this kind of pollutants. Using anion-exchange membrane chromatography, vitellogenin (VTG) from the plasma of 17β-estradiol (E2) treated Chinese rare minnow was rapidly purified within 15 min. Both polyclonal antibody (PcAb) and monoclonal antibody (McAb) against rare minnow VTG (R-VTG) were prepared in rabbit and Balb/c mice, respectively. The competitive enzyme-linked immunosorbent assays (ELISA) based on either PcAb or McAb were developed to identify and quantify R-VTG in the plasma, and these two methods showed similar characteristics. The detection limits of both assays were lower than 3 ng mL−1 with the working ranges covering three magnitudes. The recovery efficiencies of PcAb and McAb based ELISA were 104.2% and 102.6%, respectively; and the intra-assay and inter-assay of these two assays were 6.2% and 9.2%, 8.6% and 12.8%, respectively. These results indicated that the described competitive ELISA methods were sensitive and valuable tools for quantifying vitellogenin in rare minnow plasma. These methods were then applied to measure R-VTG concentrations in plasma of male fish exposed to a series of E2 concentrations. When E2 levels were less than 10 ng L−1, R-VTG levels in plasma were comparable to that in solvent control, while R-VTG levels significantly increased 15-folds and 350-folds, respectively when E2 exposure concentrations were controlled at 10 and 50 ng L−1. The high sensitivity of Chinese rare minnow to E2 was demonstrated, making it a valuable model species to study environmental estrogens.

► Vitellogenin was rapidly purified by using anion-exchange membrane chromatography.
► Polyclonal antibody and monoclonal antibody against R-VTG were prepared.
► Homologous competitive ELISA based on either PcAb or McAb were developed.
► Using these methods to measure VTG in plasma samples, comparable results were got.
► Rare minnow is an ideal model animal to study the estrogenic effects of chemicals.