Glickman J Fraser, Wu Xiang, Mercuri Robert, Illy Chantal, Bowen Benjamin R, He Yang, Sills Matthew
Novartis Institute for Biomedical Research, Summit, NJ 07901, USA.
J Biomol Screen. 2002 Feb;7(1):3-10. doi: 10.1177/108705710200700102.
New developments in detection technologies are providing a variety of biomolecular screening strategies from which to choose. Consequently, we performed a detailed analysis of both separation-based and non-separation-based formats for screening nuclear receptor ligands. In this study, time-resolved fluorescence resonance energy transfer (TR-FRET), ALPHAScreen, and time-resolved fluorescence (TRF) assays were optimized and compared with respect to sensitivity, reproducibility, and miniaturization capability. The results showed that the ALPHAScreen system had the best sensitivity and dynamic range. The TRF assay was more time consuming because of the number of wash steps necessary. The TR-FRET assay had less interwell variation, most likely because of ratiometric measurement. Both the ALPHAScreen and the TR-FRET assays were miniaturized to 8-microl volumes. Of the photomultiplier tube-based readers, the ALPHAScreen reader (ALPHAQuest) presented the advantage of faster reading times through simultaneous reading with four photomultiplier tubes.
检测技术的新进展提供了多种生物分子筛选策略以供选择。因此，我们对用于筛选核受体配体的基于分离和非基于分离的形式进行了详细分析。在本研究中，对时间分辨荧光共振能量转移（TR-FRET）、AlphaScreen和时间分辨荧光（TRF）测定法在灵敏度、重现性和小型化能力方面进行了优化和比较。结果表明，AlphaScreen系统具有最佳的灵敏度和动态范围。由于所需的洗涤步骤数量，TRF测定法更耗时。TR-FRET测定法的孔间差异较小，这很可能是由于比率测量的原因。AlphaScreen和TR-FRET测定法都被小型化为8微升体积。在基于光电倍增管的读数器中，AlphaScreen读数器（ALPHAQuest）具有通过四个光电倍增管同时读数而实现更快读数时间的优势。
