title: Development of a lipovitellin-based sandwich ELISA for quantification of vitellogenin in surface mucus and plasma of goldfish (Carassius auratus).
abstract: 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 1.82ng/mL, and the working range was 3.9-250ng/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.
authors.affiliation: Marine Life Science College, Ocean University of China, Qingdao 266003, China.
authors.forename: Jun
authors.initials: J
authors.lastname: Wang
authors.affiliation: Marine Life Science College, Ocean University of China, Qingdao 266003, China.
authors.forename: Ruihou
authors.initials: R
authors.lastname: Shan
authors.affiliation: Marine Life Science College, Ocean University of China, Qingdao 266003, China.
authors.forename: Xiaona
authors.initials: X
authors.lastname: Zhang
authors.affiliation: Marine Life Science College, Ocean University of China, Qingdao 266003, China.
authors.forename: Hua
authors.initials: H
authors.lastname: Tian
authors.affiliation: Marine Life Science College, Ocean University of China, Qingdao 266003, China. Electronic address: weiwang@ouc.edu.cn.
authors.forename: Wei
authors.initials: W
authors.lastname: Wang
authors.affiliation: Marine Life Science College, Ocean University of China, Qingdao 266003, China. Electronic address: rusg@ouc.edu.cn.
authors.forename: Shaoguo
authors.initials: S
authors.lastname: Ru
mesh_terms: D000818:Animals; D000906:Antibodies / Q000276:immunology; D015415:Biomarkers / Q000032:analysis; D015153:Blotting, Western; D004527:Egg Proteins / Q000032:analysis* / Q000276:immunology; D004591:Electrophoresis, Polyacrylamide Gel; D004785:Environmental Pollutants / Q000633:toxicity*; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D004958:Estradiol / Q000494:pharmacology; D004967:Estrogens / Q000633:toxicity*; D005260:Female; D006054:Goldfish / Q000097:blood / Q000276:immunology; D008297:Male; D009093:Mucus / Q000737:chemistry; D012680:Sensitivity and Specificity; D014819:Vitellogenins / Q000032:analysis* / Q000276:immunology
chemical_list: D000906:Antibodies; D015415:Biomarkers; D004527:Egg Proteins; D004785:Environmental Pollutants; D004967:Estrogens; D014819:Vitellogenins; D004958:Estradiol; C001549:lipovitellin
keywords: ELISA; Goldfish; Lipovitellin; Non-invasive biomarker; Surface mucus; Vitellogenin