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Analytical and Bioanalytical Chemistry

, Volume 378, Issue 3, pp 621–633 | Cite as

Development of quantitative vitellogenin-ELISAs for fish test species used in endocrine disruptor screening

  • Bente M. Nilsen
  • Karin Berg
  • Janne K. Eidem
  • Sven-Inge Kristiansen
  • François Brion
  • Jean-Marc Porcher
  • Anders Goksøyr
Original Paper

Abstract

The yolk protein precursor vitellogenin (Vtg) in plasma has proved to be a simple and sensitive biomarker for assessing exposure of fish to environmental estrogens. Within international bodies such as the Organization for Economic Cooperation and Development (OECD) work is ongoing to develop screening and testing programmes for endocrine disrupting effects of new chemicals, and in the focus of this development are the fish test species common carp (Cyprinus carpio), fathead minnow (Pimephales promelas), zebrafish (Danio rerio) and Japanese medaka (Oryzias latipes). In this study we have developed quantitative enzyme linked immunosorbent assays (ELISAs) for Vtg in common carp/fathead minnow, zebrafish and Japanese medaka. The assays were developed using a combination of monoclonal and polyclonal fish Vtg antibodies in a sandwich format, using stabilized Vtg from the test species as a standard. The carp Vtg ELISA has a working range of 1–63 ng/mL, a minimal detection limit of 0.6 ng/mL, and may also be used for quantification of Vtg in fathead minnow. In fathead minnow whole-body homogenate samples, the practical detection limit is 400 ng/mL due to the matrix effect. The zebrafish Vtg ELISA has a working range of 0.5–63 ng/mL, a minimal detection limit of 0.4 ng/mL, and a practical detection limit of 200 ng/mL in whole-body homogenate samples. The medaka Vtg ELISA has a working range of 0.25–16 ng/mL, a minimal detection limit of 0.1 ng/mL, and a practical detection limit of 125 ng/mL in whole-body homogenate samples. The intra- and inter-assay variations were below 20% for all assays. The assays were evaluated with sets of representative samples spanning the wide dynamic range of Vtg-levels found in fish exposed to environmental estrogens, and all three assays are currently undergoing international inter-laboratory validation.

Keywords

Fathead Minnow Female Fish Japanese Medaka Quantitative Amino Acid Analysis Practical Detection Limit 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

Notes

Acknowledgements

We are grateful to Dr Robert Bringolf (Iowa State University, Ames, IA, USA) for providing fathead minnows and plasma samples from E2-induced and control fathead minnows, and to Dr Muriel Mambrini (INRA, Jouy en Josas, France) for supplying the Japanese medaka. We want to thank Dr Nancy Denslow (University of Florida, Gainesville, USA), Drs Claus Kordes and Herwig O. Gutzeit (Technical University of Dresden, Germany) and Dr Charles Tyler (University of Exeter, United Kingdom) for developing monoclonal and polyclonal antibodies used in this study, and Battelle Pacific Northwest National Laboratory (USA) and Metocean Environment Inc (Japan) for supplying samples of estrogen-exposed fathead minnows and medaka, respectively. This work was supported by The Norwegian Research Council (NFR).

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Copyright information

© Springer-Verlag 2004

Authors and Affiliations

  • Bente M. Nilsen
    • 1
  • Karin Berg
    • 1
  • Janne K. Eidem
    • 1
  • Sven-Inge Kristiansen
    • 1
  • François Brion
    • 2
  • Jean-Marc Porcher
    • 2
  • Anders Goksøyr
    • 1
    • 3
  1. 1.Biosense Laboratories ASBergenNorway
  2. 2.INERIS (Institut National de l’Environnement Industriel et des Risques)Unité d’Evaluation des Risques EcotoxicologiquesVerneuil en HalatteFrance
  3. 3.Department of Molecular BiologyUniversity of BergenBergenNorway

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