DiaMetra Testosterone Saliva Elisa Kit

CLINICAL SIGNIFICANCE
Testosterone (17β-Hydroxy-4-androstene-3-one) is a steroid hormone from the androgen group. In postpubertal males, testosterone is secreted primarily by the testes with only a small amount derived from peripheral conversion of androstenedione. In adult women over 50% of serum testosterone is derived from peripheral conversion of androstenedione secreted by the adrenal and ovary, with the remainder from direct secretion of testosterone by these glands. The level of testosterone in saliva (pg/mL) is significantly lower than levels in the general circulation (ng/mL). Testosterone effects can be classified as virilizing and anabolic effects, although the distinction is somewhat artificial, as many of the effects can be considered both. Anabolic effects include growth of muscle mass and strength, increased bone density and strength, and stimulation of linear growth and bone maturation. Virilizing effects include maturation of the sex organs, and after birth (usually at puberty) a deepening of the voice, growth of the beard and axillary hair (male secondary sex characteristics). Testosterone levels decline gradually with age in men (andropause). The signs and symptoms are nonspecific, and are generally associated with aging such as loss of muscle mass and bone density, decreased physical endurance, decreased memory ability and loss of libido. In females of all ages, elevated testosterone levels can be associated with a variety of virilizing conditions, including adrenal tumors and polycystic ovarian disease.

PRINCIPLE
The Testosterone (antigen) in the sample competes with the antigenic Testosterone conjugated with horseradish peroxidase (HRP) for binding to the limited number of antibodies anti Testosterone 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 Testosterone concentration in the sample. Testosterone 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.

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