An enzyme immunoassay for the quantitative determination of free testosterone in serum and plasma.
Testosterone is a steroid hormone from the androgen group. Testosterone is primarily secreted in the testes of males and the ovaries of females although small amounts are secreted by the adrenal glands. It is the principal male sex hormone and an anabolic steroid. In both males and females, it plays key roles in health and well-being.Due to its insolubility in aqueous solutions, for the most part Testosterone circulates in the blood bound to transport proteins. Only a small percentage (< 1%) of circulating testosterone exists as unbound or free testosterone. The majority, approximately 60%, is bound to SHBG with high affinity, while the remainder is loosely bound to albumin. Both the albumin-bound and free fractions may be biologically active, while SHBG effectively inhibits testosterone action. Testosterone effects can be classified as virilizing and anabolic effects. 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. Testosterone levels decline gradually with age in men. Measurement of the free or unbound fraction of serum testosterone has been proposed as a means of estimating the physiologically bioactive hormone. Free testosterone levels are elevated in women with hyperandrogenism associated with hirsutism in the presence or absence of polycystic ovarian disease. In addition, free testosterone measurements may be more useful than total testosterone in situations where SHBG is increased or decreased (e.g. hypothyroidism and
Testosterone in the blood is bound to SHBG (60%) and in lower quantity to other proteins (for example albumin). Only the measurement of Free Testosterone (< 1% of Total Testosterone) permits the estimating of the hormone biologically active. Free Testosterone (antigen) in the sample competes with the antigenic Testosterone conjugated with horseradish peroxidase (HRP) present in the Conjugate for binding to the antibodies anti-Testosterone coated on the microplate (solid phase). The Testosterone bound to proteins does not take part to this reaction, so it is washed away during the washing step (for total Testosterone measurement the ELISA ÒTestosteroneÓ kit is available). 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 blue color that changes into yellow when the Stop Solution (H2SO4) is added. The color intensity is inversely proportional to the Free Testosterone concentration in the sample. Free Testosterone concentration in the sample is calculated through a standard curve.