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A sensitive recombinant cell-based bioluminescent assay for detection of androgen-like compounds

Nature Protocols volume 3, pages 1895–1902 (2008)Cite this article

Abstract

We report a step-by-step protocol describing how to develop and use a yeast-based bioassay for androgen-like compounds. Saccharomyces cerevisiae cells are genetically engineered to express the human androgen receptor (hAR) and the bioluminescent (BL) reporter gene luciferase (from Photinus pyralis) under the control of the androgen response element (ARE). In the presence of androgens, activated hAR binds to the ARE sequences and activates luciferase expression. After addition of D-luciferin, luciferase activity measurements can be performed, and the BL signal is proportional to the androgenic activity of the sample. Cytotoxic effects of the sample are monitored by the use of a control yeast strain that allows BL signal correction according to cell viability. After overnight culture of the recombinant strain, the assay can be accomplished in a 96-well microplate format in 1 working day with a detection limit of 0.05 nM for testosterone and intra- and interassay variability of 14% and 23%, respectively.

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Figure 1: Schematic representation of the androgen-responsive yeast.
Figure 2: Schematic of the plasmid YipLucARE.
Figure 3: Dose–response curve for testosterone.
Figure 4: Androgenic activity of human serum samples measured with the androgen-responsive strain: 10 human male serum samples (age 18–39 years), 8 female (of premenopause age) and 2 male newborn serum samples.

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Acknowledgements

The work was partly supported by the Italian Ministry for University and Research (MIUR), by Fondazione Cassa di Risparmio di Bologna, by Maj and Tor Nessling Foundation and by the Academy of Finland.

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Authors and Affiliations

  1. Department of Pharmaceutical Sciences, University of Bologna, via Belmeloro 6, Bologna, 40126, Italy

    Elisa Michelini, Luca Cevenini, Laura Mezzanotte & Aldo Roda

  2. Department of Biology, University of Turku, Turku, FI-20014, Finland

    Piia Leskinen

  3. Department of Applied Chemistry and Microbiology, University of Helsinki, PO Box 56, Helsinki, FI-00014, Finland

    Marko Virta

  4. Department of Chemistry and Bioengineering, Tampere University of Technology, PO Box 541, Tampere, FIN-33101, Finland

    Matti Karp

  5. Biosensing Competence Centre (BCC), Tampere University of Technology, PO Box 541, Tampere, Finland

    Matti Karp

Authors
  1. Elisa Michelini
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Correspondence to Aldo Roda.

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Michelini, E., Cevenini, L., Mezzanotte, L. et al. A sensitive recombinant cell-based bioluminescent assay for detection of androgen-like compounds. Nat Protoc 3, 1895–1902 (2008). https://doi.org/10.1038/nprot.2008.189

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