Development of a scintillation proximity assay binding method for the human 5-hydroxytryptamine 6 receptor using intact cells
| Autor: | Gouming Zhang, Dianne Kowal, Karen Chan, Stanley P. Nawoschik, John Dunlop, Tikva Carrick |
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| Rok vydání: | 2008 |
| Předmět: |
Biophysics
CHO Cells Ligands Biochemistry Binding Competitive law.invention HeLa Automation Radioligand Assay Cricetulus law Cricetinae Animals Humans Centrifugation Receptor Molecular Biology 5-HT receptor Chromatography biology Ligand binding assay Chinese hamster ovary cell Reproducibility of Results Cell Biology biology.organism_classification Molecular biology Serotonin Receptor Agonists Kinetics Lysergic Acid Diethylamide Scintillation proximity assay Receptors Serotonin Recombinant DNA Scintillation Counting Serotonin Antagonists Filtration Antipsychotic Agents HeLa Cells |
| Zdroj: | Analytical biochemistry. 381(1) |
| ISSN: | 1096-0309 |
| Popis: | We describe the first validated scintillation proximity assay (SPA) binding method for quantitation of 3 H-labeled d-lysergic acid diethylamide (LSD) binding to recombinant human 5-hydroxytryptamine 6 (5-HT 6 ) receptors expressed in Chinese hamster ovary (CHO)–Dukx and HeLa cells. The assay was developed using intact cells as a receptor source because membrane fractions derived from these cells failed to discern specific binding from a high level of nonspecific binding. The pharmacological binding profile of seven 5-HT 6 agonists and antagonists using intact CHO–Dukx/5-HT 6 cells in the SPA format was similar to data obtained from a filtration binding assay using HeLa/5-HT 6 membranes. K i values and rank order of potencies obtained in the SPA format were consistent with published filtration data as follows: SB-271046 ( K i = 1.9 nM) > methiothepin ( K i = 6.2 nM) > mianserin ( K i = 74.3 nM) > 5-methoxytryptamine (5-MeOT, K i = 111 nM) > 5-HT ( K i = 150 nM) > ritanserin ( K i = 207 nM) > 5-carboxamidotryptamine (5-CT, K i = 704 nM). Additional evaluation with four antipsychotics demonstrated strong agreement with previous literature reports. A high specific binding signal and low assay variability, as determined by Z ′ = 0.81 ± 0.017, make the SPA format amenable to automation and higher throughput; hence, this assay can be a viable alternative to the more labor-intensive filtration and centrifugation methods. |
| Databáze: | OpenAIRE |
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