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
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.