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A cell‐free bioassay for the detection of androgens
Author(s) -
Cooper Elliot R.,
Hughes Gillian,
Kauff Alexia,
Sutherland Emma,
Ashley Zoe,
Heather Alison K.
Publication year - 2021
Publication title -
drug testing and analysis
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.065
H-Index - 54
eISSN - 1942-7611
pISSN - 1942-7603
DOI - 10.1002/dta.3024
Subject(s) - rna , bioassay , androgen , enhancer , transcription (linguistics) , in vitro , biology , chemistry , microbiology and biotechnology , transcription factor , biochemistry , gene , genetics , hormone , linguistics , philosophy
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 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.