Testosterone is a steroid hormone with secreted from the Leydig cells of the testis in the male, adrenals and the ovaries. The dihydro derivative of Testosterone exerts a potent anabolic action responsible for the post pubescent growth rate and subsequent muscle and bone tissue maintenance of adult males. Testosterone assays are of significance in a number of endocrine dysfunction as in adult Leydig cell or seminiferous cell failure. Testosterone levels in serum may be raised by certain drugs such as 19-nortesterone, epitestosterone, ethisterone and Danazol.
The Testosterone quantitative test is based on a solid-phase enzyme immunoassay based on competitive binding method. A sample (serum/ plasma/urine) containing an unknown amount of Testosterone to be assayed (unlabeled antigen) is added to a standard amount of a conjugated Testosterone(labeled antigen). The labeled and unlabeled antigens are then allowed to compete for high affinity binding sites of anti-rabbit Testosterone antibodies on a limited number of goat anti rabbit IgG antibodies coated on to the plate. After washing away the free antigen, the amount of labeled antigen in the sample is reversibly proportional to the concentration of the unlabeled antigen. The actual concentrations in unknown samples are obtained by means of a standard curve based on known concentrations of unlabeled antigen analyzed in parallel with the unknowns. After washing, TMB substrate solution is added and the enzyme is allowed to react for a fixed time before the reaction is terminated. Absorbencies are measured at 450 nm using ELISA plate reader. A standard curve is produced using values from 5 standards from which absorbency values for blank tubes have been subtracted. Results for unknown may be read directly from this standard curve using either manual calculation or by a suitable computer program. This kit is suitable for the direct measurement of Testosterone in serum samples. It may also be used following an extraction procedure, for assaying urinary Testosterone.
<Quality Control Data>
A typical Testosterone ELISA standard curve run as a quality control of each lot is given below:
|Primate/related Testosterone concentration ng/mL||Absorbency 450nm|
< Sensitivity >
The sensitivity of the assay is 0.2 ng/mL
1. Microtiter wells coated with Goat anti rabbit IgG antibody
2. Enzyme-labeled Testosterone reagent, 12 mL
3. Rabbit anti testosterone, 7 mL
4. Testosterone reference set, 0.25mL each 0, 0.1, 0.5, 2.0, 5.0 and 20 ng/mL
5. Quality control set of 2 QC1 (<2.0ng/mL) QC2 (10ng/mL)
6. TMB Color Reagent, 12 mL
7. Stopping Solution, 6 mL
8. 20 X Wash Buffer, 20 mL.
9. Sample Diluent , 20 mL