Laboratory of Biophysics, Department of Cell Biology and Anatomy, Institute of Biomedicine, University of Turku, Turku, Finland.
Anal Chem. 2013 Feb 19;85(4):2276-81. doi: 10.1021/ac303215r. Epub 2013 Feb 6.
Two homogeneous assay systems have been combined to provide a new cell-based functional assay. The assay can be used to identify ligand binding to β(2)-adrenergic receptors, but also the downstream response can be determined in the same assay. Both the quenching resonance energy transfer (QRET) and the DiscoveRx PathHunter assay formats allow the use of intact cells. The homogeneous QRET technique is a single-label approach based on nonspecific quenching of the time-resolved luminescence, enabling agonist and antagonist receptor binding measurements. The commercial PathHunter assay is in turn based on enzyme fragment complementation, which can be detected on the basis of chemiluminescence signal. In the PathHunter technology the enzyme complementation is recorded immediately downstream of agonist-induced receptor activation. The new multiparametric detection technology combines these two assay methods enabling the identification of agonist, and antagonist binding to the receptor, and the agonist-induced response. Using the QRET and the PathHunter methods a panel of β(2)-adrenergic receptor ligands (epinephrine, terbutaline, metaproterenol, salmeterol, propranolol, alprenolol, bisoprolol, ICI 118,551, and bucindolol) was tested to prove the assay performance. The signal-to-background ratio for tested ligands ranged from 5 to 11 and from 6 to 18 with QRET and PathHunter, respectively. Combined homogeneous assay technique can provide an informative method for screening purposes and an efficient way to monitor receptor-ligand interaction, thus separating agonist from antagonist.
两种均相测定系统已被结合，以提供一种新的基于细胞的功能测定法。该测定法可用于鉴定配体与β(2)-肾上腺素能受体的结合，但也可以在同一测定中确定下游反应。均相 QRET 技术和 DiscoveRx PathHunter 测定格式都允许使用完整细胞。基于时间分辨荧光的非特异性猝灭的单一标记方法，可进行激动剂和拮抗剂受体结合测量。商用 PathHunter 测定法基于酶片段互补，可基于化学发光信号进行检测。在 PathHunter 技术中，酶互补在激动剂诱导的受体激活后立即记录。新的多参数检测技术结合了这两种测定方法，能够识别激动剂和拮抗剂与受体的结合，以及激动剂诱导的反应。使用 QRET 和 PathHunter 方法，测试了一组β(2)-肾上腺素能受体配体（肾上腺素、特布他林、间羟异丙肾上腺素、沙美特罗、普萘洛尔、阿替洛尔、比索洛尔、ICI 118,551 和布新洛尔），以证明该测定法的性能。用 QRET 和 PathHunter 检测的测试配体的信号背景比分别为 5-11 和 6-18。组合的均相测定技术可为筛选目的提供一种信息丰富的方法，并且是监测受体-配体相互作用的有效方法，从而将激动剂与拮抗剂分离。
