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Cortisol assay

Cortisol, also known as hydrocortisone, is the major glucocorticoid produced and secreted by the adrenal cortex. It is present in blood as free protein or bound to cortisol binding protein (CBG) and 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 11β-hydroxysteroid dehydrogenase (type 1 or 2). Based on our patented HTRF® technology, 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 11β-HSD1 activity (native, recombinant or microsomal).

Features

  • Detection in complex samples such as serum and whole cells
  • Low cross-reactivity with cortisone
  • Signal to background ratio >25
  • Highly sensitive assay with large dynamic range
  • Adapted to 96, 384 and 1536-well formats
  • Amenable to automation and HTS

Applications

  • Cortisol measurement
  • 11β-HSD1 screening

The cortisol assay is a monoclonal antibody-based competitive assay requiring a single 2 hour incubation following cell stimulation. The assay can also be run with purified 11β-HSD1 enzyme.

 

The basic assay is run in two steps:

Stimulation step:

cells (or microsomes) are stimulated with compounds

Detection step:

cortisol levels are measured by successive addition of d2-labeled cortisol and a Cryptate-labeled anti-cortisol MAb. Fluorescence is read after a 2 hour incubation.

Sample compatibility: serum, whole cells, & liver microsomes
Detection limit: 70 pg/mL (2 fmol cortisol/well)
Dynamic range 0.1 to 100 ng/mL
S/B > 30
Cross-reactivity: Cortisone 0.9%
Corticosterone 7.0%
Prednisolone 5.7%
Cortisone 0.9%
Progesterone 2.7%
Prednisone < 0.3%
Testosterone < 0.002%
DHEA < 0.02%
Dexamethasone< 0.0007%
Z': 0.9 for 20 µl assay volumes

A typical standard curve is shown. The assay was performed in a final 20µl assay volume in a low volume 384-well plate

Cisbio Bioassays's cortisol assay is based on our robust and patented HTRF technology. One advantage of HTRF 's robustness is the ability to use samples such as serum, cells and microsome preparations. Figure 2 summarizes the results of a microsomal 11β-HSD1 assay performed using HTRF cortisol reagents in low-volume 384-well plates. Known inhibitors of 11β-HSD1, Carbenoxolone and Glycyrrhetinic acid, blocked the conversion of cortisone into cortisol. IC50 values were 0.33 and 0.31µM respectively.

Microsomal 11β-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 cortisol-d2 conjugate and Eu3+ Cryptate labeled anti-cortisol antibody added. Plates were incubated at RT for 4 hours after reagent additions and emissions read at 620 and 665nm.

Ordering Info

DescriptionCat. noProduct insertMSDS
Cortisol kit - 1,000 tests62CRTPEB
Cortisol kit - 20,000 tests62CRTPEC

Companion products

DescriptionCat. noProduct insertMSDS
Cortisol kit calibrator62CORCDA
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Cortisol kit control62CORTDA
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Diluent 1 - 20 ml62DL1DDD
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Reconstitution buffer 2 - 13 ml62RB2RDD
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