title: Development of a 5-hydroxytryptamine(2A) receptor binding assay for high throughput screening using 96-well microfilter plates.
abstract: A high throughput screening method for the analysis of 5-hydroxytryptamine(2A) (5-HT(2A)) receptor binding parameters has been developed, using 96-well filter plates of the Millipore MultiScreen system in combination with a MicroBeta PLUS microplate scintillation counter. MAFB filter plates (GF/B filter over a Durapore membrane) were used because of the lower nonspecific binding of the radioligand to GF/B filter material than to GF/C filters. Comparing different scintillation cocktails, highest counting efficiency and shortest equilibration time were detected with Betaplatescint, after drying the plates at 50 degrees C for 2 h. Measuring the plates without the plastic underdrain increased the counting efficiency by about 39% as compared with counting the plate with the underdrain intact. Presoaking the wells with 0.5% polyethyleneimine for 2 h reduced the nonspecific binding to the filter material by about 50%. A linear relationship of protein concentration and radioligand binding was established up to a protein concentration of 165 microg of protein/well. In the assays, 70 microg of protein/well was generally used, which has turned out to be favorable with respect to the number of counts obtained. When a higher concentration of protein was used, the period of time needed to aspirate the plate was too long because of obstruction of the filter material. Receptor-radioligand equilibration was reached after about 20 min at concentrations less than 0.05 nM [(3)H]ketanserin-HCl; at higher concentrations it was reached after about 10 min. Saturation analysis of [(3)H]ketanserin-HCl resulted in a mean B(max) of 393 fmol/mg protein and a K(D) of 2.0 nM using rat frontal cortex as a receptor source. Competition experiments with known 5-HT(2A) receptor ligands-DOB-HCl (K(i) = 59 nM), DOET-HCl (K(i) = 137 nM), DOM-HCl (K(i) = 533 nM), DMT (K(i) = 1,985 nM), and TMA-HCl (K(i) = 22,340 nM)-were in accordance with literature values.
authors.affiliation: Pharmaceutical Institute, University of Tübingen, Tübingen, Germany.
authors.forename: A
authors.initials: A
authors.lastname: Harms
authors.forename: D
authors.initials: D
authors.lastname: Gündisch
authors.forename: C E
authors.initials: CE
authors.lastname: Müller
authors.forename: K A
authors.initials: KA
authors.lastname: Kovar
mesh_terms: D000818:Animals; D001667:Binding, Competitive; D001921:Brain / Q000378:metabolism; D004353:Drug Evaluation, Preclinical / Q000295:instrumentation / Q000379:methods*; D066298:In Vitro Techniques; D007650:Ketanserin / Q000378:metabolism; D007700:Kinetics; D011869:Radioligand Assay / Q000295:instrumentation / Q000379:methods*; D051381:Rats; D044402:Receptor, Serotonin, 5-HT2A; D011985:Receptors, Serotonin / Q000378:metabolism*; D012588:Scintillation Counting / Q000295:instrumentation / Q000379:methods
chemical_list: D044402:Receptor, Serotonin, 5-HT2A; D011985:Receptors, Serotonin; D007650:Ketanserin