Publication Details

Category Text Publication
Reference Category Journals
DOI 10.1016/S1532-0456(01)00194-6
Title (Primary) 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
Author Fenske, M.; van Aerle, R.; Brack, S.; Tyler, C.R.; Segner, H.
Source Titel Comparative Biochemistry and Physiology C-Toxicology & Pharmacology
Year 2001
Department OEC; COE
Volume 129
Page From 217
Page To 232
Language englisch
Keywords Enzyme-linked immunosorbent assay (ELISA); Environmental estrogens; 17α-Ethinylestradiol (EE2); Homologous antibody; Vitellogenin (VTG); Zebrafish
Abstract Vitellogenin (VTG) was isolated by anion exchange chromatography from plasma of female zebrafish (Danio rerio) induced with 17α-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) ≤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.
Persistent UFZ Identifier https://www.ufz.de/index.php?en=20939&ufzPublicationIdentifier=6311
Fenske, M., van Aerle, R., Brack, S., Tyler, C.R., Segner, H. (2001):
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
Comp. Biochem. Physiol. C-Toxicol. Pharmacol. 129 , 217 - 232 10.1016/S1532-0456(01)00194-6