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Development Assay for of a 5-Hydroxytryptamine2A Receptor Binding High Throughput Screening Using 96-Well Microfilter PlatesPharmaceutical Institute, University of Tilbingen, Tubingen, Germany
Pharmaceutical Institute, University of Bonn, Bonn, Germany
Pharmaceutical Institute, University of Bonn, Bonn, Germany
Pharmaceutical Institute, University of Tilbingen, Tubingen, Germany A high throughput screening method for the analysis of 5-hydroxytryptamine2A (5-HT2A) 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°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 µg of protein/well. In the assays, 70 µg 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 [3H]ketanserin-HCl; at higher concentrations it was reached after about 10 min. Saturation analysis of [3H]ketanserin-HCI resulted in a mean Bmax of 393 fmol/mg protein and a KD of 2.0 nM using rat frontal cortex as a receptor source. Competition experiments with known 5-HT2A receptor ligands-DOB-HCI (Ki = 59 nM), DOET-HCl (Ki = 137 nM), DOM-HCl (Ki = 533 nM), DMT (K; = 1,985 nM), and TMA-HCl (Ki = 22,340 nM)-were in accordance with literature values.
Journal of Biomolecular Screening, Vol. 5, No. 4,
269-277 (2000) |
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