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Multiparametric Homogeneous Method for Identification of Ligand Binding to G Protein-Coupled Receptors: Receptor-Ligand Binding and beta-Arrestin Assay
Tekijät: Kopra K, Kainulainen M, Mikkonen P, Rozwandowicz-Jansen A, Hanninen P, Harma H
Kustantaja: AMER CHEMICAL SOC
Julkaisuvuosi: 2013
Journal: Analytical Chemistry
Tietokannassa oleva lehden nimi: ANALYTICAL CHEMISTRY
Lehden akronyymi: ANAL CHEM
Numero sarjassa: 4
Vuosikerta: 85
Numero: 4
Aloitussivu: 2276
Lopetussivu: 2281
Sivujen määrä: 6
ISSN: 0003-2700
DOI: https://doi.org/10.1021/ac303215r
Tiivistelmä
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 beta(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 Path Hunter 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 Path Hunter assay is in turn based on enzyme fragment complementation, which can be detected on the basis of chemiluminescence signal. In the Path Hunter 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 Path Hunter methods a panel of beta(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 Path Hunter, 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.
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 beta(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 Path Hunter 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 Path Hunter assay is in turn based on enzyme fragment complementation, which can be detected on the basis of chemiluminescence signal. In the Path Hunter 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 Path Hunter methods a panel of beta(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 Path Hunter, 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.
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