Establishment and application of multiple immunoassays for environmental estrogens based on recombinant Japanese flounder (Paralichthys olivaceus) choriogenin protein.

ABSTRACT

Environmental estrogens have generated great concern because of their potential threat to aquatic organisms; however, the commonly used vitellogenin (Vtg) biomarker detection methods are not capable of detecting estrogenic activity below 10 ng/L 17ß-estradiol. In this study, we developed multiple immunoassays based on Japanese flounder (Paralichthys olivaceus) choriogenin (Chg), a highly sensitive biomarker of environmental estrogens. Chg genes (ChgL and ChgH) of Japanese flounder were cloned for the first time, and a recombinant ChgL protein with a molecular weight of approximately 52 kDa was prepared using a prokaryotic expression system and purified using Ni-affinity column chromatography. Subsequently, specific monoclonal antibodies against ChgL were prepared and used to develop sandwich enzyme-linked immunosorbent assays (ELISAs), which had a detection range of 3.9-250 ng/mL and detection limit of 1.9 ng/mL. An immunofluorescence method was also established and used to visually detect ChgL induction in the tissues. In addition, a lateral flow immunoassay for ChgL that could detect estrogen activity within 10 min was developed. Finally, the reliability of the immunoassays was examined by measuring ChgL induction in the plasma and tissues of Japanese flounder exposed to 0, 2, 10, and 50 ng/L 17α-ethinylestradiol (EE2). The results showed that 2 ng/L EE2 notably increased ChgL levels in the plasma, demonstrating that ChgL is more sensitive than Vtg to environmental estrogens; 50 ng/L EE2 induced obvious Chg induction in the sinusoidal vessels of the liver. Conclusions taken together, this study provides reliable methods for sensitive and rapid detection of estrogenic activity in aquatic environments.