title: Development of a scintillation proximity assay binding method for the human 5-hydroxytryptamine 6 receptor using intact cells.
abstract: 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.
authors.affiliation: Neuroscience Discovery Research, Wyeth Research, Princeton, NJ 08543, USA. carrict@wyeth.com
authors.forename: Tikva
authors.initials: T
authors.lastname: Carrick
authors.forename: Dianne
authors.initials: D
authors.lastname: Kowal
authors.forename: Stanley
authors.initials: S
authors.lastname: Nawoschik
authors.forename: Gouming
authors.initials: G
authors.lastname: Zhang
authors.forename: Karen
authors.initials: K
authors.lastname: Chan
authors.forename: John
authors.initials: J
authors.lastname: Dunlop
mesh_terms: D000818:Animals; D014150:Antipsychotic Agents / Q000032:analysis / Q000378:metabolism; D001331:Automation; D001667:Binding, Competitive / Q000187:drug effects; D016466:CHO Cells; D006224:Cricetinae; D003412:Cricetulus; D005374:Filtration; D006367:HeLa Cells; D006801:Humans; D007700:Kinetics; D008024:Ligands; D008238:Lysergic Acid Diethylamide / Q000032:analysis / Q000378:metabolism; D011869:Radioligand Assay / Q000379:methods*; D011985:Receptors, Serotonin / Q000378:metabolism*; D015203:Reproducibility of Results; D012588:Scintillation Counting; D012702:Serotonin Antagonists / Q000032:analysis / Q000378:metabolism; D017366:Serotonin Receptor Agonists / Q000032:analysis / Q000378:metabolism
chemical_list: D014150:Antipsychotic Agents; D008024:Ligands; D011985:Receptors, Serotonin; D012702:Serotonin Antagonists; D017366:Serotonin Receptor Agonists; C097216:serotonin 6 receptor; D008238:Lysergic Acid Diethylamide