Development of a 5-hydroxytryptamine(2A) receptor binding assay for high throughput screening using 96-well microfilter plates

A high throughput screening method for the analysis of 5-hydroxytryptamine( 2A) (5-HT2A) receptor binding parameters has been developed, using 96-well filter plates of the Millipore MultiScreen system in combination with a Mic roBeta PLUS microplate scintillation counter. MAFB filter plates (GF/B filt er over a Durapore membrane) were used because of the lower nonspecific bin ding of the radioligand to GF/B filter material than to GF/C filters, Compa ring different scintillation cocktails, highest counting efficiency and sho rtest equilibration time were detected with Betaplatescint, after drying th e 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 r adioligand binding was established up to a protein concentration of 165 mu g of protein/well. In the assays, 70 mu g of protein/well was generally use d, which has turned out to be favorable with respect to the number of count s 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 th e filter material. Receptor-radioligand equilibration was reached after abo ut 20 min at concentrations less than 0.05 nM [M-3]ketanserin-HCl; at highe r concentrations it was reached after about 10 min, Saturation analysis of [H-3]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 exp eriments with known 5-HT2A 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.