Development and validation of a homologous zebrafish (Danio rerio Hamilton-Buchanan) vitellogenin enzyme-linked immunosorbent assay (ELISA) and its application for studies on estrogenic chemicals.
Vitellogenin (VTG) was isolated by anion exchange chromatography from plasma of female zebrafish (Danio rerio) induced with 17alpha-ethinylestradiol (EE2). The purity of the VTG isolate was confirmed by polyacrylamide gel electrophoresis (SDS-PAGE). Purified VTG was used to raise polyclonal antibodies in rabbits and the specificity of the antisera for VTG confirmed by Western blot analysis of plasma proteins separated by SDS-PAGE. The antibodies cross-reacted with two proteins in the plasma of female zebrafish, with molecular masses of approximately 142 and 171 kDa. No cross-reactivity was observed with any other plasma proteins. A competitive enzyme-linked immunosorbent assay (ELISA) was developed using the polyclonal zebrafish VTG (z-VTG) antibodies and purified z-VTG as ligand and standard, respectively. The z-VTG ELISA was sensitive with a detection limit of between 2.0 and 3.0 ng purified VTG/ml, and a working range between 3 and 500 ng/ml (30-85% binding). The ELISA demonstrated precision, with inter- and intra-assay variations of 7.5+/-2.7 and 4.9+/-1.4%, respectively. Plasma from adult zebrafish and whole body homogenates from juvenile zebrafish diluted parallel with the z-VTG standard in the ELISA, validating the assay for quantifying z-VTG in both of these tissues. Exposure of adult male zebrafish to EE2 via water induced a concentration-dependent induction of VTG with a lowest observed effect concentration (LOEC) < or =1.67 ng EE2/l (for a 21-day exposure). The homologous z-VTG ELISA provides a valuable tool for the study of environmental estrogens in zebrafish.
Fenske
M
M
Department of Chemical Ecotoxicology, UFZ Center for Environmental Research, Leipzig-Halle GmbH, Permoserstrasse 15, 04318, Leipzig, Germany. mf@uoe.ufz.de
van Aerle
R
R
Brack
S
S
Tyler
C R
CR
Segner
H
H
D000818:Animals; D001798:Blood Proteins / Q000096:biosynthesis; D004305:Dose-Response Relationship, Drug; D004781:Environmental Exposure / Q000032:analysis*; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods; D045166:Estradiol Congeners / Q000494:pharmacology; D004997:Ethinyl Estradiol / Q000494:pharmacology; D005260:Female; D008297:Male; D015203:Reproducibility of Results; D014819:Vitellogenins / Q000096:biosynthesis*; D015027:Zebrafish / Q000378:metabolism*
D001798:Blood Proteins; D045166:Estradiol Congeners; D014819:Vitellogenins; D004997:Ethinyl Estradiol