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Assay Method Information

Assay Name:  Binding Affinity Assay (Ki) and cAMP Accumulation Activity Assay (EC50)
Description:  The affinities of test compounds for human V2 receptor were evaluated by the radioligand binding study using membrane fractions isolated from CHO cells expressing human V2 receptors. 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). 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). EC50 values were determined as the concentration of the test compound required to increase the cAMP level to 50% of the maximum response to AVP. All assays were performed in triplicate.
Affinity data for this assay
 

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Last update November 1, 2007
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