A1 Refereed original research article in a scientific journal
A homogeneous single-label time-resolved fluorescence cAMP assay
Authors: Martikkala E, Rozwandowicz-Jansen A, Hänninen P, Petäjä-Repo U, Härmä H
Publication year: 2011
Journal: Journal of Biomolecular Screening
Journal name in source: Journal of Biomolecular Screening
Number in series: 3
Volume: 16
Issue: 3
First page : 356
Last page: 362
Number of pages: 7
ISSN: 1087-0571
DOI: https://doi.org/10.1177/1087057110397356
Web address : http://api.elsevier.com/content/abstract/scopus_id:79954586393
Abstract
G-protein-coupled receptors (GPCRs) are an important class of pharmaceutical drug targets. Functional high-throughput GPCR assays are needed to test an increasing number of synthesized novel drug compounds and their function in signal transduction processes. Measurement of changes in the cyclic adenosine monophosphate (cAMP) concentration is a widely used method to verify GPCR activation in the adenylyl cyclase pathway. Here, a single-label time-resolved fluorescence and high-throughput screening (HTS)-feasible method was developed to measure changes in cAMP levels in HEK293
cells overexpressing either ß
-adrenergic or δ-opioid receptors. In the quenching resonance energy transfer (QRET) technique, soluble quenchers reduce the signal of unbound europium(III)-labeled cAMP in solution, whereas the antibody-bound fraction is fluorescent. The feasibility of this homogeneous competitive assay was proven by agonist-mediated stimulation of receptors coupled to either the stimulatory G
or inhibitory G
proteins. The reproducibility of the assays was excellent, and Z values exceeded 0.7. The dynamic range, signal-to-background ratio, and detection limit were compared with a commercial time-resolved fluorescence resonance energy transfer (TR-FRET) assay. In both homogeneous assays, similar assay parameters were obtained when adenylyl cyclase was stimulated directly by forskolin or via agonist-mediated activation of the G
-coupled ß
AR. The advantage of using the single-label approach relates to the cost-effectiveness of the QRET system compared with the two-label TR-FRET assay as there is no need for labeling of two binding partners leading to reduced requirements for assay optimization. (Journal of Biomolecular Screening. 2011:16:356-362) © 2011 Society for Laboratory Automation and Screening.
G-protein-coupled receptors (GPCRs) are an important class of pharmaceutical drug targets. Functional high-throughput GPCR assays are needed to test an increasing number of synthesized novel drug compounds and their function in signal transduction processes. Measurement of changes in the cyclic adenosine monophosphate (cAMP) concentration is a widely used method to verify GPCR activation in the adenylyl cyclase pathway. Here, a single-label time-resolved fluorescence and high-throughput screening (HTS)-feasible method was developed to measure changes in cAMP levels in HEK293
cells overexpressing either ß
-adrenergic or δ-opioid receptors. In the quenching resonance energy transfer (QRET) technique, soluble quenchers reduce the signal of unbound europium(III)-labeled cAMP in solution, whereas the antibody-bound fraction is fluorescent. The feasibility of this homogeneous competitive assay was proven by agonist-mediated stimulation of receptors coupled to either the stimulatory G
or inhibitory G
proteins. The reproducibility of the assays was excellent, and Z values exceeded 0.7. The dynamic range, signal-to-background ratio, and detection limit were compared with a commercial time-resolved fluorescence resonance energy transfer (TR-FRET) assay. In both homogeneous assays, similar assay parameters were obtained when adenylyl cyclase was stimulated directly by forskolin or via agonist-mediated activation of the G
-coupled ß
AR. The advantage of using the single-label approach relates to the cost-effectiveness of the QRET system compared with the two-label TR-FRET assay as there is no need for labeling of two binding partners leading to reduced requirements for assay optimization. (Journal of Biomolecular Screening. 2011:16:356-362) © 2011 Society for Laboratory Automation and Screening.
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