Development of a lipovitellin-based sandwich ELISA for quantification of vitellogenin in surface mucus and plasma of goldfish (Carassius auratus)

• Anti-Lv antibody recognized more epitopes of purified Vtg and Lv than anti-Vtg.
• A sandwich ELISA for goldfish Vtg was developed using purified Lv and anti-Lv.
• Using Lv instead of Vtg as the standard strengthened the robustness of the ELISA.
• The Lv-based ELISA was used to quantify Vtg in surface mucus and plasma of goldfish.
• Mucus Vtg of male goldfish may be a reliable indicator for estrogenic contamination.

Goldfish (Carassius auratus) vitellogenin (Vtg) is an efficient biomarker for estrogen contamination in aquatic environments. In this study, Vtg and lipovitellin (Lv) were purified from the plasma of 17β-estradiol (E2)-induced male goldfish and unfertilized eggs of females, and were used to generate polyclonal antibodies against Vtg (anti-Vtg) and Lv (anti-Lv), respectively. SDS-PAGE and Western blot were performed to confirm the specificity of the two antibodies and the immunological similarity between Vtg and Lv. As anti-Lv recognized more antigen epitopes than anti-Vtg, it was used to develop a sandwich enzyme-linked immunosorbent assay (ELISA) for goldfish Vtg with purified Lv as the standard. The detection limit of the assay was 1.82 ng/mL, and the working range was 3.9–250 ng/mL. The use of Lv instead of Vtg as the standard provided greater precision and strengthened the robustness of the sandwich ELISA. Western blot and the Lv-based ELISA were used to detect Vtg inductions in surface mucus and plasma of E2-induced goldfish. The surface mucus Vtg level in E2-induced males was significantly higher than that in the control males and E2-induced females, and was much closer to the plasma Vtg level in E2-induced males than that in E2-induced females. Therefore, the surface mucus Vtg level of male goldfish may be a reliable indicator of estrogenic activity in the aquatic environment.