DiaMetra Androstenedione Saliva Elisa Kit

CLINICAL SIGNIFICANCE
Androstenedione (also known as Δ 4- androstenedione) is a steroid hormone produced in the adrenal glands and the gonads as an intermediate step in the biochemical pathway that produces the androgen testosterone and the estrogens estrone and estradiol. It is the common precursor of male and female sex hormones. Some androstenedione is also secreted into the plasma, and may be converted in peripheral tissues to testosterone and estrogens. Androstenedione has relatively weak androgenic activity, estimated at ~ 20% of testosterone. Secretion and production rates also exceed those of testosterone in women in whom significant extraadrenal conversion of androstenedione to testosterone occurs. In premenopausal women the adrenal glands and ovaries each produce about half of the total androstendione (about 3 mg/day). After menopause androstenedione production is about halved, primarily due to the reduction of steroid secreted by the ovary. Nevertheless, androstenedione is the principal steroid produced by the postmenopausal ovary. The high serum-saliva correlation for androstenedione suggests that individual differences in serum androstenedione levels may be accurately estimated using saliva as a non-invasive alternative specimen.

PRINCIPLE
The Androstenedione (antigen) in the sample competes with the antigenic Androstenedione conjugated with horseradish peroxidase (HRP) for binding to the limited number of antibodies anti Androstenedione 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 Androstenedione concentration in the sample. Androstenedione concentration in the sample is calculated through a calibration curve.

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.
Addition of the TMB Substrate solution initiates a kinetic reaction, which is terminated by the addition of the Stop Solution. Therefore, the TMB Substrate and the Stop Solution should be added in the same sequence to eliminate any time deviation during the reaction.

QUALITY CONTROL
Each laboratory should assay controls at normal, high and low levels range of Androstenedione 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.

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 pg/mL.