Estrogen pollution in marine environments has become a research hotspot due to its adverse effects on the reproduction of wild organisms. To early detection of estrogen pollution, this study developed two methods for detecting Japanese flounder vitellogenin (Vtg), a sensitive biomarker for environmental estrogens. Firstly, monoclonal antibodies (mAb) specific to Vtg were prepared using purified lipovitellin (Lv), a main Vtg-derived yolk protein. Anti-Lv mAb (C1F1) had the highest titer (1:256,000) and was labeled with fluorescein isothiocyanate to establish a direct immunofluorescence (DIF) method for histological detection of Vtg in tissues. Additionally, using the purified Lv and mAb, an enzyme-linked immunosorbent assay (ELISA) was developed and this assay had a detection limit of 0.75 ng/mL and a working range of 1.95-250 ng/mL. Furthermore, Vtg induction in the plasma of Japanese flounder exposed to 17β-estradiol (E2), 17α-ethinylestradiol (EE2), and bisphenol A (BPA) were quantified by ELISA, and Vtg induction in the liver of EE2-exposed Japanese flounder were measured by DIF. Finally, the distribution of Vtg in Japanese flounder was detected using these two methods. The results revealed that Vtg mainly appeared in the terminal tail fin, liver, kidney, intestine, and spleen. Considering the high concentration of Vtg and easy sample collection, the terminal tail fin could be a new alternative to plasma for Vtg quantification, while kidney and liver are suitable for histological detection of Vtg.
Keywords: Biomarker; Direct immunofluorescence; Environmental estrogens; Lipovitellin; Paralichthys olivaceus.
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