A cell‐free bioassay for the detection of androgens

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.