Kool Jeroen, van Marle André, Hulscher Saskia, Selman Maurice, van Iperen Dick J, van Altena Klaas, Gillard Michel, Bakker Remko A, Irth Hubertus, Leurs Rob, Vermeulen Nico P E
LACDR-Division of Molecular Toxicology, Department of Chemistry and Pharmaceutical Sciences, Vrije Universiteit Amsterdam, The Netherlands.
J Biomol Screen. 2007 Dec;12(8):1074-83. doi: 10.1177/1087057107308881.
A flow-through fluorescence polarization (FP) detection system that makes use of a novel high-performance liquid chromatography (HPLC) fluorescence detector modified with polarization filters was developed. This flow-through FP detection system was evaluated by using a novel and very cost-effective bioassay for cyclic adenosine monophosphate (cAMP). The bioassay was first evaluated and optimized in an FP plate reader format and subsequently in a flow-through bioassay setup. The principle of the bioassay is based on the competition of cAMP and a fluorescent cAMP derivative for the cAMP binding domain of protein kinase A. cAMP could accurately be determined over a range of 0.8 to 30 pmol/well in the plate reader FP assay and over a range of 0.3 to 50 pmol/well in the flow-through FP assay setup. High Z' factors (i.e., 0.89 for the plate reader and 0.93 for the flow-through FP cAMP assay, respectively) indicated robust assays. Finally, functional cAMP signaling of the human histamine H(3) G-protein-coupled receptor (GPCR) in cell cultures was measured with both assay formats with good sensitivities and assay windows. The pEC(50) values obtained in both assay formats were in accordance with those obtained with standard methods. The flow-through FP detection system could thus be used as a cost-effective alternative to FP plate reader assays. Moreover, the novel flow-through FP detection system for cAMP constitutes a good analytical tool to be used in the GPCR research field as an alternative to the use of FP plate readers or radioactive laboratories nowadays used for cAMP measurements.
开发了一种流通式荧光偏振（FP）检测系统，该系统利用了一种经偏振滤光片修饰的新型高效液相色谱（HPLC）荧光检测器。通过使用一种新型且成本效益极高的环磷酸腺苷（cAMP）生物测定法对该流通式FP检测系统进行了评估。该生物测定法首先在FP酶标仪形式下进行评估和优化，随后在流通式生物测定装置中进行评估。该生物测定法的原理基于cAMP和一种荧光cAMP衍生物对蛋白激酶A的cAMP结合结构域的竞争。在酶标仪FP测定中，cAMP在0.8至30 pmol/孔的范围内能够准确测定，在流通式FP测定装置中，cAMP在0.3至50 pmol/孔的范围内能够准确测定。高Z'因子（即酶标仪为0.89，流通式FP cAMP测定分别为0.93）表明测定方法稳健。最后，使用这两种测定形式对细胞培养物中人类组胺H(3) G蛋白偶联受体（GPCR）的功能性cAMP信号进行了测量，灵敏度和测定窗口良好。两种测定形式获得的pEC(50)值与使用标准方法获得的值一致。因此，流通式FP检测系统可以作为FP酶标仪测定的一种经济有效的替代方法。此外，用于cAMP的新型流通式FP检测系统构成了一种良好的分析工具，可在GPCR研究领域中用作替代目前用于cAMP测量的FP酶标仪或放射性实验室的工具。
