A cell-free bioassay for the detection of androgens.

Autor: Cooper ER; Department of Physiology, School of Biomedical Sciences, University of Otago, Dunedin, New Zealand., Hughes G; Department of Physiology, School of Biomedical Sciences, University of Otago, Dunedin, New Zealand.; InsituGen Ltd, Dunedin, New Zealand., Kauff A; Department of Physiology, School of Biomedical Sciences, University of Otago, Dunedin, New Zealand.; InsituGen Ltd, Dunedin, New Zealand., Sutherland E; Department of Physiology, School of Biomedical Sciences, University of Otago, Dunedin, New Zealand., Ashley Z; Department of Physiology, School of Biomedical Sciences, University of Otago, Dunedin, New Zealand., Heather AK; Department of Physiology, School of Biomedical Sciences, University of Otago, Dunedin, New Zealand.; InsituGen Ltd, Dunedin, New Zealand.
Jazyk: angličtina
Zdroj: Drug testing and analysis [Drug Test Anal] 2021 May; Vol. 13 (5), pp. 903-915. Date of Electronic Publication: 2021 Mar 28.
DOI: 10.1002/dta.3024
Abstrakt: Androgens remain abused performance-enhancing drugs in sports. Technologies based on mass spectrometry can detect all forms of androgens but fail if the androgen represents a novel structure. A bioassay detects androgens based on function rather than structure. To date, there has been limited adoption of cell-based in vitro bioassays as a screening tool for nontargeted androgen detection because they require expert personnel and specialized equipment to perform. We now describe the development of a cell-free version of an androgen in vitro bioassay. Stage 1 involved in vitro transcription/translation reactions (IVTT) using a DNA template encoding an enhancer/androgen response element (ARE) regulatory region upstream of a minimal promoter that drives expression of a reporter protein. The assay detected testosterone across the concentration range of 106.7 to 0.0144 ng/ml (3.7 × 10 -7 to 5 × 10 -11 M), with an EC 50 of 6.63 ng/ml (23 nM). To reduce complexity, Stages 2-4 of development included just in vitro transcription (IVT) reactions, whereby the output was an RNA molecule. Stage 2 involved directly labelling the RNA molecule with fluorophore-labelled nucleotide triphosphates, Stage 3 involved reverse transcription-polymerase chain reaction (PCR) of the RNA molecule, and Stage 4 utilized an RNA aptamer, Mango II, as its RNA output. The Stage 4 product detected testosterone across the range of 106.7-0.0001 ng/ml (3.7 × 10 -7 to 5 × 10 -13 M), with an EC 50 of 0.04 ng/ml (0.155 nM). Further to this, we show that the Stage 4 product can detect other androgenic molecules. Relative to cell-based bioassays, the Stage 4 product is easy to perform and could be developed into a routine, high-throughput, nontargeted androgen screen.
(© 2021 John Wiley & Sons, Ltd.)
Databáze: MEDLINE
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