A Cell-Free Bioassay for the Detection of Androgens

A Cell-Free Bioassay for the Detection of Androgens / Elliot R. Cooper, Gillian Hughes, Alexia Kauff, Emma Sutherland, Zoe Ashley, Alison K. Heather. - (Drug Testing and Analysis (2021) 11 March)

  • PMID: 33709622
  • DOI: 10.1002/dta.3024


Abstract

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 non-targeted 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/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 (3.7X10-7 -5X10-11 M), with an EC50 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 labeling the RNA molecule with fluorophore-labeled nucleotide triphosphates, stage 3 involved reverse transcription-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 (3.7X10-7 -5X10-13 M), with an EC50 of 0.04 ng/mL (0.155 nM). Further to this, we showed that the stage 4 product could 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, non-targeted androgen screen.

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Science
Study
Date
11 March 2021
People
Ashley, Zoe
Cooper, Elliot R.
Heather, Alison K.
Hughes, Gillian
Kauff, Alexia
Sutherland, Emma
Country
New Zealand
Language
English
Other organisations
University of Otago
Analytical aspects
Testing method development
Doping classes
S1. Anabolic Agents
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18 March 2021
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20 March 2021
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