PUBLICATION
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
- Authors
- Fenske, M., van Aerle, R., Brack, S., Tyler, C.R., and Segner, H.
- ID
- ZDB-PUB-010807-24
- Date
- 2001
- Source
- Comparative biochemistry and physiology. Toxicology & pharmacology : CBP 129(3): 217-232 (Journal)
- Registered Authors
- Fenske, Martina
- Keywords
- enzyme-linked immunosorbent assay; (ELISA); environmental estrogens; 17a-ethinylestradiol (EE2); homologous anti-body; vitellogenin (VTG); zebrafish
- MeSH Terms
-
- Animals
- Blood Proteins/biosynthesis
- Dose-Response Relationship, Drug
- Environmental Exposure/analysis*
- Enzyme-Linked Immunosorbent Assay/methods
- Estradiol Congeners*/pharmacology
- Ethinyl Estradiol/pharmacology
- Female
- Male
- Reproducibility of Results
- Vitellogenins/biosynthesis*
- Zebrafish/metabolism*
- PubMed
- 11461838 Full text @ Comp. Biochem. Physiol. C Toxicol. Pharmacol.
Citation
Fenske, M., van Aerle, R., Brack, S., Tyler, C.R., and 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. Comparative biochemistry and physiology. Toxicology & pharmacology : CBP. 129(3):217-232.
Abstract
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)
Genes / Markers
Expression
Phenotype
Mutations / Transgenics
Human Disease / Model
Sequence Targeting Reagents
Fish
Orthology
Engineered Foreign Genes
Mapping