title: Fathead minnow (Pimephales promelas) vitellogenin: purification, characterization and quantitative immunoassay for the detection of estrogenic compounds.
abstract: The egg yolk precursor protein, vitellogenin (VTG), was purified from blood plasma of 17beta-estradiol (E2)-treated male fathead minnows (Pimephales promnelas) by anion-exchange chromatography on DEAE-agarose. A rabbit antiserum was raised against their blood plasma and then adsorbed with plasma from untreated (control) males to render the antiserum specific to VTG. The adsorbed antiserum was used to detect fathead minnow VTG (fVTG) in Western and dot blotting experiments and in an enzyme-linked immunosorbent assay (ELISA). The antiserum recognised fVTG as a approximately 156 kDa protein in plasma from vitellogenic females and E2-injected males but not untreated males. Its identity was confirmed by analysis of: (1) amino acid composition; (2) an internal amino acid sequence; (3) reactivity to the homologous antiserum; and (4) recognition by monoclonal antibodies prepared against the VTG from common carp (Cyprinus carpio) and brown bullhead (Ameiurus nebulosus). Specificity of the homologous antiserum to fVTG was confirmed by Western blotting of serially diluted plasma from vitellogenic females. Utility of the antiserum and purified fVTG for detecting exposure of male fathead minnows to estrogenic compounds was verified using a dot blotting immunoassay of fVTG and detected by chemiluminescence. Adult male fish were exposed to various concentrations of E2 (10(-8), 10(-9) and 10(-10) M) in their rearing water and plasma assayed for the presence of VTG at different time points (2, 7, 14 and 21 days). A competitive, antibody-capture, quantitative ELISA was then developed based on the purified fVTG and its respective antiserum. The ELISA was validated by demonstrating parallel binding slopes of dilution curves prepared with plasma from E2-injected males, vitellogenic females, and aqueous egg extracts as compared with purified fVTG standard. Plasma concentrations of VTG as low as 3 ng ml(-1) were detected in the ELISA, for which inter- and intra-assay coefficients of variation were both less than 5%. Furthermore, plasma from control males was unreactive with the fVTG antiserum. The VTG ELISA could be useful for the detection of estrogenic properties associated with certain compounds and could be easily incorporated into standard laboratory toxicity assays using this species.
authors.affiliation: Department of Toxicology, North Carolina State University, Raleigh 27695, USA.
authors.forename: L G
authors.initials: LG
authors.lastname: Parks
authors.forename: A O
authors.initials: AO
authors.lastname: Cheek
authors.forename: N D
authors.initials: ND
authors.lastname: Denslow
authors.forename: S A
authors.initials: SA
authors.lastname: Heppell
authors.forename: J A
authors.initials: JA
authors.lastname: McLachlan
authors.forename: G A
authors.initials: GA
authors.lastname: LeBlanc
authors.forename: C V
authors.initials: CV
authors.lastname: Sullivan
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D015153:Blotting, Western; D002852:Chromatography, Ion Exchange; D003530:Cyprinidae; D004784:Environmental Monitoring; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D004967:Estrogens / Q000032:analysis*; D008297:Male; D008969:Molecular Sequence Data; D015203:Reproducibility of Results; D014819:Vitellogenins / Q000096:biosynthesis / Q000737:chemistry / Q000302:isolation & purification*
chemical_list: D004967:Estrogens; D014819:Vitellogenins