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 plasm a of female zebrafish (Danio rerio) induced with 17 alpha -ethinylestradiol (EE2). The purity of the VTG isolate was confirmed by polyacrylamide gel e lectrophoresis (SDS-PAGE). Purified VTG was used to raise polyclonal antibo dies in rabbits and the specificity of the antisera for VTG confirmed by We stern blot analysis of plasma proteins separated by SDS-PACE. The antibodie s cross-reacted with two proteins in the plasma of female zebrafish, with m olecular masses of approximately 142 and 171 kDa. No cross-reactivity was o bserved with any other plasma proteins. A competitive enzyme-linked immunos orbent assay (ELISA) was developed using the polyclonal zebrafish VTG (z-VT G) 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% bindin g). The ELISA demonstrated precision, with inter- and intra-assay variation s of 7.5 +/- 2.7 and 4.9 +/- 1.4%, respectively. Plasma from adult zebrafis h and whole body homogenates from juvenile zebrafish diluted parallel with the z-VTG standard in the EI-ISA, validating the assay for quantifying z-VT G in both of these tissues. Exposure of adult male zebrafish to EE2 via wat er induced a concentration-dependent induction of VTG with a lowest observe d effect concentration (LOEC) less than or equal to 1.67 ng EE2/1 (for a 21 -day exposure). The homologous z-VTG ELISA provides a valuable tool for the study of environmental estrogens in zebrafish. (C) 2001 Elsevier Science I nc. All rights reserved.