DiaMetra Urinary Cortisol Elisa Kit

CLINICAL SIGNIFICANCE
Cortisol is a steroid hormone released from the adrenal cortex in response to an hormone called ACTH (produced by the pituitary gland), it is involved in the response to stress; it increases blood pressure, blood sugar levels, may cause infertility in women, and suppresses the immune system.
Cortisol acts through specific intracellular receptors and has effects in numerous physiologic systems, including immune function, glucose-counter regulation, vascular tone, substrate utilization and bone metabolism. Cortisol is excreted primarily in urine in an unbound (free) form.
Cortisol is bound, in plasma, from corticosteroid-binding globulin (CBG, transcotin), with high affinity, and from albumin. Only free cortisol is available to most receptors. These normal endogenous functions are the basis for the physiological consequences of chronic stress - prolonged cortisol secretion causes muscle wastage, hyperglycaemia, and suppresses immune / inflammatory responses. The same consequences arise from long-term use of glucocorticoid drugs.
The free cortisol fraction represents the metabolically active cortisol. In normal conditions, less than 1% is excreted in urine. In pathological conditions (Cushing syndrome), free urinary cortisol levels are very high as excess plasma cortisol doesn’t not bind to the CBG, so is excreted in urine.
During pregnancy or estro-progestogen treatment an increase of plasmatic cortisol caused by an increment of the production of the transport protein, but the levels of free urinary cortisol results normal to indicate a correct adrenal function.

PRINCIPLE OF THE METHOD
The Urinary 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.
Cortisol concentration in the sample is calculated through a calibration curve.

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 calibrators are stable at 2 – 8°C for 6 months.
Once opened, the conjugate is at 2 – 8°C for 6 months.
The diluted wash solution is stable for 30 days at 2 – 8°C.

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 controls provided in the kit should be tested as unknowns 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.

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. Other curve fitting algorithms are not recommended.
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.
To calculate the cortisol concentration in urine, calculate as above and correct for total volume of volume of urine collected in 24 hours:
                                              ng/mL x vol (mL) urine 24h/ 1000 = μg Cortisol/24h