DiaMetra Cortisol Elisa Kit

CLINICAL SIGNIFICANCE 
Cortisol is a glucocorticoid steroid hormone synthesised from cholesterol in the zona fasciculata of the adrenal cortex. Glucocorticoids are predominantly stress hormones that regulate a variety of physiological processes by binding to glucocorticoid receptors expressed throughout the body1 . Endogenous glucocorticoids like cortisol play a crucial role in establishing the body’s homeostasis after physical or psychological stress. Additionally, cortisol plays a role in the metabolism of carbohydrates, fat and protein, the maintenance of myocardial function2 and is involved in the development and function of the central nervous system, the immune system and the gastrointestinal system3 .

PRINCIPLE OF THE METHOD
The Cortisol ELISA is a competitive enzyme immunometric assay (ELISA) where Cortisol (antigen) in the sample competes with the antigenic Cortisol conjugated with horseradish peroxidase (HRP) for binding to the limited number of antibodies anti-Cortisol coated on the microplate (solid phase). After the incubation, the bound/free separation is performed by a simple solid phase washing. Then, the enzyme HRP in the bound fraction reacts with the Substrate (H2O2) and the TMB Substrate and develops a blue colour that changes into yellow when the Stop Solution (H2SO4) is added. The colour intensity is inversely proportional to the Cortisol concentration in the sample.

QUALITY CONTROL
Good Laboratory Practice (GLP) requires the use of quality control specimens in each series of assays in order to check the performance of the assay. Controls should be treated as unknown samples, and the results analysed with appropriate statistical methods. The kit control provided in the kit should be tested as unknown and are intended to assist in assessing the validity of results obtained with each assay plate. The mean concentration of each control level is documented in the QC report included with each kit. These mean concentration levels are determined over several assays which are run in duplicate in multiple locations across each plate. DiaMetra recommends the users to maintain graphic records of the control values generated with each assay run, including the running means, SDs and %CVs. This information will facilitate the controls trending analysis relating to the performance of current and historical control lots relative to the supplied Quality Control data. The trending will assist in the identification of assays which give control values significantly different from their average range.

REAGENT STORAGE AND STABILITY
Store the kit at 2 – 8°C in the dark.
The kit is stable at 2 – 8°C until the expiry date stated on the external kit label.
Once opened, the kit is stable at 2 – 8°C for 5 months.
The diluted wash solution is stable for 30 days at 2-8°C.
Important note: open the bag containing the Coated Microplate only when it is at room temperature and close it immediately after use.

CALCULATION OF RESULTS
A variety of data reduction software packages are available, which may be employed to generate the mean calibration curve and to calculate the mean concentrations of unknown samples and controls. A 4-parameter logistic (4PL) curve fit, including Calibrator 0 is required. Alternatively, a calibration curve may be prepared on semilog graph paper by plotting mean absorbance on the Y-axis against concentration of analyte on the X-axis. Calibrator 0 should be included in the calibration curve. Read the mean absorbance value of each unknown sample off the curve. In order for the assay results to be considered valid the kit calibrators and control must fall within the specifications detailed in the lot specific certificate of analysis. If a control is out of its specified range, the associated test results are invalid and samples must be retested.

QUALITY CONTROL
Good Laboratory Practice (GLP) requires the use of quality control specimens in each series of assays in order to check the performance of the assay. Controls should be treated as unknown samples, and the results analysed with appropriate statistical methods.
The kit control provided in the kit should be tested as unknown and are intended to assist in assessing the validity of results obtained with each assay plate. The mean concentration of each control level is documented in the QC report included with each kit.
These mean concentration levels are determined over several assays which are run in duplicate in multiple locations across each plate. DiaMetra recommends the users to maintain graphic records of the control values generated with each assay run, including the running means, SDs and %CVs.
This information will facilitate the controls trending analysis relating to the performance of current and historical control lots relative to the supplied Quality Control data. The trending will assist in the identification of assays which give control values significantly different from their average range.