Cooper Elliot R, Hughes Gillian, Kauff Alexia, Sutherland Emma, Ashley Zoe, Heather Alison K
Department of Physiology, School of Biomedical Sciences, University of Otago, Dunedin, New Zealand.
InsituGen Ltd, Dunedin, New Zealand.
Drug Test Anal. 2021 May;13(5):903-915. doi: 10.1002/dta.3024. Epub 2021 Mar 28.
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 to 5 × 10 M), with an EC 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 to 5 × 10 M), with an EC 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.
雄激素在体育界仍然是被滥用的提高成绩的药物。基于质谱分析的技术可以检测所有形式的雄激素，但如果雄激素具有新的结构，这些技术就会失效。生物测定法是根据功能而非结构来检测雄激素的。迄今为止，基于细胞的体外生物测定法作为非靶向雄激素检测的筛选工具的应用有限，因为它们需要专业人员和专门设备来操作。我们现在描述一种无细胞版雄激素体外生物测定法的开发。第一阶段涉及使用DNA模板进行体外转录/翻译反应（IVTT），该DNA模板编码一个增强子/雄激素反应元件（ARE）调控区域，该区域位于驱动报告蛋白表达的最小启动子上游。该测定法在106.7至0.0144 ng/ml（3.7×10至5×10 M）的浓度范围内检测到睾酮，其半数有效浓度（EC）为6.63 ng/ml（23 nM）。为了降低复杂性，开发的第二至第四阶段仅包括体外转录（IVT）反应，其输出结果是一个RNA分子。第二阶段涉及用荧光团标记的三磷酸核苷酸直接标记RNA分子，第三阶段涉及RNA分子的逆转录-聚合酶链反应（PCR），第四阶段利用一种RNA适体Mango II作为其RNA输出。第四阶段的产物在106.7-0.0001 ng/ml（3.7×10至5×10 M）的范围内检测到睾酮，其EC为0.04 ng/ml（0.155 nM）。此外，我们表明第四阶段的产物可以检测其他雄激素分子。相对于基于细胞的生物测定法，第四阶段的产物易于操作，可以开发成一种常规的、高通量的、非靶向的雄激素筛查方法。
