| Assay Method Information | |
| | Homogeneous Time-Resolved Fluorescence (HTRF) Assay |
| Description: | In vitro inhibition of 11beta -HSD1 was determined with HTRF (Homogeneous Time-Resolved Fluorescence) technology (cisbio international, France) detecting cortisolgenerated from cortisterone by human liver microsomes. Compounds were incubated for 1 hour at 37 C. in Tris buffer (20 mM tris, 5 mM EDTA, pH 6.0) containing NADPH (200 uM) and cortisone (80 nM). Cortisol generated in the reaction was then detected by a competitive immunoassay involving two HTRF conjugates: cortisol linked to XL665 and anticortisol antibody labeled with Europium cryptate. The incubation period for detection reaction was 2 hours. The amount of cortisol is determined by reading the time-resolved fluorescence of the wells (Ex 320/75 nm; Em 615/8.5 nm and 665/7.5 nm). The ratio of the two emission signals was then calculated (Em665*10000/Em615). Each assay contained incubations with vehicle controls instead of compound as controls for non-inhibited cortisol generation. |
| Affinity data for this assay | |
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