DiaMetra DHEA-S Saliva Elisa Kit

CLINICAL SIGNIFICANCE
endogenous natural steroid hormone with 19 carbon atoms. It is the principal steroid hormone produced by the secretion of the adrenal glands, but it is also produced in the gonads and brain. DHEA is the most abundant circulating steroid in human beings.
DHEA-S is a natural steroid hormone found primarily in the kidneys and it is derived from the enzymatic conversion of DHEA in the adrenal and extra-adrenal tissues. It is the most abundant hormone in the human body and is a 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: for example its production in the brain suggests a role as neurosteroid.
The majority of DHEA-S in saliva is non-protein bound and enters the saliva via intracellular mechanisms. Salivary DHEA-S levels are unaffected by salivary flow rate or salivary enzymes. Measurement of serum DHEA-S is a useful marker of adrenal androgen synthesis. Abnormally low levels may occur in have been reported in hypoadrenalism, while elevated levels occur in several conditions, e.g. virilizing adrenal adenoma and carcinoma, 21- hydroxylase and 3β-hydroxysteroid dehydrogenase deficiencies and in some cases of female hirsutism. Women with polycystic ovary syndrome tend to have normal or mildly elevated levels of DHEAS.

PRINCIPLE
The 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 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 blu color that changes into yellow when the Stop Solution (H2SO4) is added. The colour intensity is inversely proportional to the DHEA-S concentration of in the sample. DHEA-S concentration in the sample is calculated through a calibration curve.

QUALITY CONTROL
Each laboratory should assay controls at normal, high and low levels range of DHEA-S for monitoring assay performance. These controls should be treated as unknowns and values determined in every test procedure performed. Quality control charts should be maintained to follow the performance of the supplied reagents. Pertinent statistical methods should be employed to ascertain trends. The individual laboratory should set acceptable assay performance limits. Other parameters that should be monitored include the 80, 50 and 20% intercepts of the calibration curve for run-to-run reproducibility. In addition, maximum absorbance should be consistent with past experience. Significant deviation from established performance can indicate unnoticed change in experimental conditions or degradation of kit reagents. Fresh reagents should be used to determine the reason for the variations.

PRECAUTIONS
Please adhere strictly to the sequence of pipetting steps provided in this protocol. The performance data represented here were obtained using specific reagents listed in this Instruction For Use.
All reagents should be stored refrigerated at 2-8°C in their original container. Any exceptions are clearly indicated. The reagents are stable until the expiry date when stored and handled as indicated.
Allow all kit components and specimens to reach room temperature (22-28°C) and mix well prior to use.
Do not interchange kit components from different lots. The expiry date printed on box and vials labels must be observed. Do not use any kit component beyond their expiry date.
If you use automated equipment, the user has the responsibility to make sure that the kit has been appropriately tested.
The incomplete or inaccurate liquid removal from the wells could influence the assay precision and/or increase the background. To improve the performance of the kit on automatic systems is recommended to increase the number of washes.
It is important that the time of reaction in each well is held constant for reproducible results. Pipetting of samples should not extend beyond ten minutes to avoid assay drift. If more than 10 minutes are needed, follow the same order of dispensation. If more than one plate is used, it is recommended to repeat the dose response curve in each plate.
 

RESULTS
Mean Absorbance 
Calculate the mean of the absorbance (Em) for each point of the calibration curve (C0-C4) and of each sample.
Calibration curve 
Plot the mean value of absorbance (Em) of the Calibrators (C0-C4) against concentration. Draw the best-fit curve through the plotted points. (es: Four Parameter Logistic).
Calculation of Results
Interpolate the values of the samples on the calibration curve to obtain the corresponding values of the concentrations expressed in ng/mL.