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

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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.

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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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Authors and Affiliations

  1. Biosense Laboratories AS, Thormøhlens gt. 55, 5008 , Bergen, Norway

    Bente M. Nilsen, Karin Berg, Janne K. Eidem, Sven-Inge Kristiansen & Anders Goksøyr

  2. INERIS (Institut National de l’Environnement Industriel et des Risques), Unité d’Evaluation des Risques Ecotoxicologiques, BP2, 60550 , Verneuil en Halatte, France

    François Brion & Jean-Marc Porcher

  3. Department of Molecular Biology, University of Bergen, HIB, P.O. Box 7800, 5020 , Bergen, Norway

    Anders Goksøyr

Authors
  1. Bente M. Nilsen
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  2. Karin Berg
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  3. Janne K. Eidem
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  4. Sven-Inge Kristiansen
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  5. François Brion
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  6. Jean-Marc Porcher
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  7. Anders Goksøyr
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Correspondence to Bente M. Nilsen.

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Nilsen, B.M., Berg, K., Eidem, J.K. et al. Development of quantitative vitellogenin-ELISAs for fish test species used in endocrine disruptor screening. Anal Bioanal Chem 378, 621–633 (2004). https://doi.org/10.1007/s00216-003-2241-2

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  • DOI: https://doi.org/10.1007/s00216-003-2241-2

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