title: Preparation of a polyclonal antibody against goldfish (Carassius auratus) vitellogenin and its application to detect the estrogenic effects of monocrotophos pesticide.
abstract: Goldfish (Carassius auratus) represents a good model to detect the estrogenic effects of chemicals, and vitellogenin (Vtg) is a vital indicator of estrogenic activity. The heterologous anti-carp Vtg antibody has previously been used for goldfish Vtg detection. Here, we report the preparation of an anti-goldfish Vtg antibody to improve the sensitivity and specificity of goldfish Vtg immunoassays. Vtg was purified from the plasma of 17β-estradiol (E2)-induced goldfish by gel filtration followed by anion-exchange chromatography. It was characterized as a phospholipoglycoprotein with an apparent molecular weight of ~460 kDa and separated into three major polypeptides corresponding to ~130, ~106, and ~81 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). A polyclonal antibody against goldfish Vtg was raised in rabbits and found to be specific for goldfish Vtg through immunoelectrophoresis and Western blot. A sensitive sandwich enzyme-linked immunosorbent assay (ELISA) was developed for the quantification of plasma Vtg, with a detection limit of 3.6 ng/mL and a detection range from 7.8 to 250 ng/mL. The intra- and inter-assay coefficients of variations were 2.4-6.8% and 6.7-10.8%, respectively. Additionally, we qualitatively and quantitatively detected the induction of Vtg in male fish exposed to 0.01, 0.01, and 1.00 mg/L monocrotophos pesticide by Western blot and ELISA. The homologous sandwich ELISA based on the anti-goldfish Vtg antibody could provide a valuable tool for the study of estrogenic effects of exogenous chemicals on goldfish.
authors.affiliation: Marine Life Science College, Ocean University of China, 5 Yushan Road, Qingdao 266003, Shandong Province, China.
authors.forename: Jun
authors.initials: J
authors.lastname: Wang
authors.affiliation: Marine Life Science College, Ocean University of China, 5 Yushan Road, Qingdao 266003, Shandong Province, China.
authors.forename: Xin
authors.initials: X
authors.lastname: Bing
authors.affiliation: Marine Life Science College, Ocean University of China, 5 Yushan Road, Qingdao 266003, Shandong Province, China.
authors.forename: Kun
authors.initials: K
authors.lastname: Yu
authors.affiliation: Marine Life Science College, Ocean University of China, 5 Yushan Road, Qingdao 266003, Shandong Province, China.
authors.forename: Hua
authors.initials: H
authors.lastname: Tian
authors.affiliation: Marine Life Science College, Ocean University of China, 5 Yushan Road, Qingdao 266003, Shandong Province, 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, 5 Yushan Road, Qingdao 266003, Shandong Province, China. Electronic address: rusg@ouc.edu.cn.
authors.forename: Shaoguo
authors.initials: S
authors.lastname: Ru
mesh_terms: D000818:Animals; D000906:Antibodies / Q000276:immunology*; D000918:Antibody Specificity; D015153:Blotting, Western; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods; D004967:Estrogens / Q000633:toxicity*; D029941:Fish Proteins / Q000737:chemistry / Q000276:immunology* / Q000302:isolation & purification; D006054:Goldfish / Q000097:blood / Q000276:immunology; D008297:Male; D008999:Monocrotophos / Q000633:toxicity*; D010575:Pesticides / Q000633:toxicity*; D011817:Rabbits; D014819:Vitellogenins / Q000097:blood / Q000737:chemistry / Q000276:immunology* / Q000302:isolation & purification
chemical_list: D000906:Antibodies; D004967:Estrogens; D029941:Fish Proteins; D010575:Pesticides; D014819:Vitellogenins; D008999:Monocrotophos
keywords: ELISA; Goldfish; Monocrotophos.; Polyclonal antibody; Vitellogenin