Cisbio´s HTRF® cortisol assay performed on the PHERAstar

Abstract

Cortisol is a corticosteroid hormone present in many metabolic processes, inducing key enzymes of carbohydrate, fat and protein metabolism. Cortisol also acts as an anti-inflammatory and immunosuppressor. One way to create this important hormone is by the reduction of cortisone by NADPH dependent 11 betahydroxysteroid dehydrogenase type 1 (11 beta-HSD1).1 This enzyme can be found in several tissues but it is mostly present in liver and fat cells.2 Recently Cisbio developed an assay to determine the activity of 11 beta-HSD1 using HTRF® technology. HTRF® (Homogeneous Time-Resolved Fluorescence) is based on TR-FRET (time-resolved fluorescence resonance energy transfer), a combination of FRET chemistry and the use of fluorophores with long emission halflives (Europium, Eu3+). FRET uses two fluorophores, a donor and an acceptor. Excitation of the donor by an energy source (e.g. flash lamp or laser) triggers an energy transfer to the acceptor if they are within a given proximity to each other. The acceptor in turn emits light at a given wavelength.3 BMG LABTECH’s PHERAstar (Figure 1) is a multifunctional plate reader that combines rapid plate reading necessary for high throughput screening (HTS) with the enhanced performance and sensitivity needed to read small fluid volumes. The PHERAstar has been designed to read all HTS detection modes (fluorescence intensity, time-resolved fluorescence, fluorescence polarization, luminescence, AlphaScreenTM and absorption) in all plate formats up to 1536 wells. To meet the HTRF® requirements the PHERAstar uses a unique HTRF®-specific optical module that can measure two emission signals simultaneously. Assay Principle