| Assay Method Information | |
| | Binding Assay for Human V1a Receptor |
| Description: | Chinese hamster ovary (CHO) cells stably expressing human V1a receptors. Cells were washed with phosphate buffered saline, and then collected in ice-cold hypotonic buffer. Subsequently, cells were collected using a cell scraper and then homogenized using POLYTRON followed by centrifugation at 4 deg C. The supernatant was centrifuged (35,000g, 30 min) at 4 deg C, and the pellet was suspended in Tris buffer as membrane fractions. The affinities of test compounds for human V1a receptor were evaluated by the radioligand binding study. For the competitive binding study, drug compound solution and [3H]vasopressin were mixed with membrane suspension in reaction buffer. This mixture was incubated at room temperature for 60 min. Reactions were terminated by filtration through UniFilter GF/B (Perkin-Elmer) using a MicroMate Cell Harvester (Packard Instrument Company, Meriden, CT, USA). The radioactivity retained on the filter was counted by TopCountTM microplate scintillation counter (Perkin-Elmer) using the scintillation cocktail (MicroScinti-40TM, Perkin-Elmer). Nonspecific binding or total binding were determined by including 1 uM AVP or without test compounds in the reaction mixture, respectively. Specific binding was calculated as total binding minus nonspecific binding. The concentration of each compound required to reduce specific binding of [3H]vasopressin by 50% (IC50 value) was obtained by non-linear regression analysis. A Kd value of [3H]vasopressin for each vasopressin receptor was yielded by Scatchard plot analysis. The affinity constants (Ki values) were calculated from the following equation, using the Kd values yielded from each separate experiment. Ki =IC50/(1 + [[3H]vasopressin concentration]/Kd). |
| Affinity data for this assay | |
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