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首页> 外文期刊>Analytical chemistry >Multiparametric Homogeneous Method for Identification of Ligand Binding to G Protein-Coupled Receptors: Receptor-Ligand Binding and β-Arrestin Assay
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Multiparametric Homogeneous Method for Identification of Ligand Binding to G Protein-Coupled Receptors: Receptor-Ligand Binding and β-Arrestin Assay

机译：用于鉴定配体与G蛋白偶联受体结合的多参数均相方法：受体-配体结合和β-arrestin分析

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摘要

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.
机译：两种同类的检测系统已经结合在一起，可以提供一种新的基于细胞的功能检测方法。该测定法可用于鉴定与β_2-肾上腺素能受体结合的配体，但也可在同一测定法中确定下游反应。猝灭共振能量转移（QRET）和DiscoveRx PathHunter分析方法都允许使用完整细胞。均质QRET技术是基于时间分辨发光的非特异性淬灭的单标记方法，可进行激动剂和拮抗剂受体结合测量。商业PathHunter测定法又基于酶片段互补，可以基于化学发光信号进行检测。在PathHunter技术中，紧随激动剂诱导的受体激活的下游记录了酶的互补作用。新的多参数检测技术结合了这两种测定方法，可鉴定激动剂，拮抗剂与受体的结合以及激动剂诱导的应答。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，异丙肾上腺素，沙美特罗，普萘洛尔，a烯醇，比索洛尔，ICI 118,551和丁星醇），以证明测定性能。使用QRET和PathHunter进行测试的配体的信噪比分别为5至11和6至18。联合的均相测定技术可提供用于筛选目的的信息方法，以及监测受体-配体相互作用的有效方法，从而将激动剂与拮抗剂分离。

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