Diametra DHEA-S Elisa Kit

CLINICAL SIGNIFICANCE
Dehydroepiandrosterone sulfate (DHEA-S), is a natural steroid hormone found atop of the kidneys in the human body. DHEA-S derived from enzymatic conversion of DHEA in adrenal and extradrenal tissues. DHEA-S is also produced in the gonads, adipose tissue and the brain. It is the most abundant hormone in the human body and it is precursor of all sex steroids. As most DHEA-S is produced by the zona reticularis of the adrenal, it is argued that there is a role in the immune and stress response. DHEA-S may have more biologic roles. Its production in the brain suggests that is also has a role as a neurosteroid.

PRINCIPLE OF THE METHOD
The DHEA-S ELISA is a competitive enzyme immunometric assay (ELISA) where DHEA-S (antigen) in the sample competes with the antigenic DHEA-S conjugated with horseradish peroxidase (HRP) for binding to the limited number of antibodies anti- DHEA-S 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 DHEA-S concentration in the sample.
DHEA-S concentration in the sample is calculated through a calibration curve.

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. A smoothed spline fit including Calibrator 0 can be used. 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.

Preparation of Samples
The determination of DHEA-S can be performed in human serum or plasma samples. 
All serum and plasma samples must be pre-diluted with sample diluent 1:50; for example, 20 µL of sample may be diluted with 980 µL of serum diluent. 
Collect blood by venepuncture into vacutainers and separate serum (after clot formation) or plasma from the cells by centrifugation. 
Store the sample at -20°C if the determination is not performed on the same day of the sample collection. Before using, mix gently, for 5 minutes, with a roller mixer.

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.