A cell‐free bioassay for the detection of androgens. Issue 5 (28th March 2021)

• Record Type: Journal Article
• Authors: Cooper, Elliot R.; Hughes, Gillian; Kauff, Alexia; Sutherland, Emma; Ashley, Zoe; Heather, Alison K.
• Other Names: Cawley Adam guestEditor.
• Is Part Of: Drug testing and analysis. Volume 13:Issue 5(2021)
• Page Start: 903
• Page End: 915
• Publication Date: 2021-03-28
• Subjects: androgens -- bioassay -- in vitro transcription -- sports doping; Drugs -- Analysis -- Periodicals; Drug testing -- Periodicals; Chemistry, Forensic -- Periodicals; 615.1901
• DOI: 10.1002/dta.3024
• Languages: English
• ISSNs: 1942-7603

Abstract

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 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 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 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 EC50 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. Abstract : In vitro bioassays detect androgens based on biological function rather than structure, thus, could act as a nontargeted screening tool. We have developed a cell‐free in vitro androgen bioassay that could be developed into a routine, high‐throughput test.