Description
Cortisol, also known as hydrocortisone, is the major glucocorticoid produced and secreted by the adrenal cortex. It is involved in regulation of glucose metabolism, fat degradation, blood pressure levels, and stress inhibition. Cortisol also diffuses into cells, where its level is highly regulated by 11ß-hydroxysteroid dehydrogenase (type 1 or 2). The cortisol assay allows for rapid and accurate cortisol measurement, even in complex samples such as liver microsomes, whole cells, and animal serum. This assay can also be used to assess 11ß-HSD1 activity (native, recombinant, or microsomal).
HTRF assays offer many advantages over other technologies:
Homogeneous add-and-read format
No wash steps
Low background
Straightforward miniaturization from 96- or 384-well microplates to high density assay formats such as 384-well low volume and 1536-well plates
Stable signal, providing flexibility in time of readout or size of assays
Assay principle
The kit is based on a competitive format involving a specific antibody labelled with Cryptate (donor) and cortisol coupled to d2 (acceptor). Cortisol produced by cells competes with d2-labelled cortisol for binding to monoclonal anti-cortisol Eu Cryptate.
Assay protocol
The cortisol assay features a streamlined protocol with only two incubation steps:
- Cell stimulation by target compounds
- Cortisol detection using HTRF reagents.
This protocol requires only a single, two-hour incubation period following cell stimulation.
Cortisol assessment on microsomes
The cortisol kit is intended to accurately measure hydrocortisone in complex samples such as serum, cells and microsome preparations. Figure 2 summarizes the results of a microsomal 11ß-HSD1 assay performed using HTRF cortisol reagents in low-volume 384-well plates. Known inhibitors of 11ß-HSD1, Carbenoxolone and Glycyrrhetinic acid, blocked the conversion of cortisone into cortisol. IC50 values were 0.33 and 0.31 µM respectively.
Microsomal 11ß-HSD1 (0.2mg/mL) was stimulated by NADPH (200µM) and cortisone (160nM) in a 384 low-volume plate. Various concentrations of the Glycyrrhetinic acid and Carbenoxolene inhibitors were added.
A standard curve with known cortisol concentrations was run concomitantly. Plates were incubated for 2 hours at 37°C and then a cortisol-d2 conjugate and Eu3+ Cryptate labeled anti-cortisol antibodies were added.
Plates were incubated at RT for 4 hours after reagent additions and emissions read at 620 and 665 nm.
