Enzyme Immunoassay for the Quantitative Determination of Testosterone Concentration in Human Serum

for in vitro diagnostic use

Product Description
Testosterone (17β-hydroxyandrost-4-ene-3-one) is a C19 steroid with an unsaturated bond between C-4 and C-5, a ketone group in C-3 and a hydroxyl group in the β position at C-17. This steroid hormone has a molecular weight of 288.4.

Testosterone is the most important androgen secreted into the blood. In males, testosterone is secreted primarily by the Leydig cells of the testes; in females ca. 50% of circulating testosterone is derived from peripheral conversion of androstenedione, ca. 25% from the ovary and ca. 25% from the adrenal glands.

Testosterone is responsible for the development of secondary male sex characteristics and its measurements are helpful in evaluating the hypogonadal states.

In women, high levels of testosterone are generally found in hirsutism and virilization, polycystic ovaries, ovarian tumors, adrenal tumors and adrenal hyperplasia.

In men, high levels of testosterone are associated to the hypothalamic pituitary unit diseases, testicular tumors, congenital adrenal hyperplasia and prostate cancer.

Low levels of testosterone can be found in patients with the following diseases: Hypopituitarism, Klinefelter’s syndrome, Testicular feminization, Orchidectomy and Cryptorchidism, enzymatic defects and some autoimmune diseases. The Testosterone EIA kits are designed for the measurement of total Testosterone in human serum.

Principle
The Testosterone EIA is based on the principle of competitive binding between Testosterone in the test specimen and Testosterone-HRP conjugate for a constant amount of rabbit anti-Testosterone. In the incubation, goat anti-rabbit IgG-coated wells are incubated with 10µl of Testosterone standards, controls, patient samples, 100µl Testosterone-HRP conjugate reagent and 50µl rabbit anti-Testosterone reagent at 37°C for 90 minutes.
During the incubation, a fixed amount of HRP-labeled Testosterone competes with the endogenous Testosterone in the standard, sample, or quality control serum for a fixed number of binding sites of the specific Testosterone antibody.

Thus, the amount of Testosterone peroxidase conjugate immunologically bound to the well progressively decreases as the concentration of Testosterone in the specimen increases. Unbound Testosterone peroxidase conjugate is then removed and the wells washed. Next, a solution of TMB Reagent is then added and incubated at room temperature for 20 minutes, resulting in the development of blue color.
The color development is stopped with the addition of 1N HCl, and the absorbance is measured spectrophotometrically at 450nm. The intensity of the color formed is proportional to the amount of enzyme present and is inversely related to the amount of unlabeled Testosterone in the sample.
A standard curve is obtained by plotting the concentration of the standard versus the absorbance. The Testosterone concentration of the specimens and controls run concurrently with the standards can be calculated from the standard curve.

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