Free Testosterone ELISA
The Calbiotech, Inc. (CBI) Free Testosterone ELISA Kit is intended for the measurement of Free Testosterone in serum or 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. Testosterone is the principal male sex hormone, is an anabolic steroid and, for both males and females, plays a key role in health and well-being. Testosterone within the circulation is principally bound to proteins, the most important of which, is sex hormone binding globulin (SHBG). Measurement of the free or unbound fraction of serum testosterone has been proposed as a mean 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 obesity).
The CBI Testosterone ELISA kit 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-Free Testosterone. In the incubation, goat anti-rabbit IgG-coated wells are incubated with 25μl of Testosterone standards, patient samples, 50μl testosterone-HRP conjugate reagent and 50μl rabbit anti-free testosterone reagent at room temperature for 60 minutes. During the incubation, a fixed amount of HRP labeled testosterone competes with the endogenous testosterone in the standard and sample, for a fixed number of binding sites of the specific free testosterone antibody. Thus, the amount of testosterone peroxidase conjugate immunologically bound to the well progressively decreases as the concentration of free testosterone in the specimen increases. Unbound testosterone peroxidase conjugate is then removed and the wells washed. Next, 100Il of TMB Reagent is added and incubated at room temperature for 15 minutes, resulting in the development of blue color. The color development is stopped with the addition of 50Il stop solution, and the absorbance is measured spectrophotometrically at 450nm. A standard curve is prepared relating color intensity to the concentration of the Free Testosterone.
- McCann D, Kirkish L. Evaluation of Free Testosterone in serum. J.Clin. Immunoassay 1985; 8:234-236.
- Ekins R.P. Free hormones in blood J. Clin. Immunoassay 1984; 7(2): 163-180.
- Paulson JD, et al. Free Testosterone concentration in serum: elevation is the hallmark of hirsutism. Am.J.Obst. Gynecol 1977; 128:851-857.
- Odlind V. et al. Plasma androgenic acitivity in women with acne vulgaris and in healthy gils before, during and after puberty. Clin.Endocrinology 1982; 16:243-249.
- Green PJ. Free Testosterone determination by ultrafiltration and comparison with dialysis.Clin.Chem. 1982;28:163-180.
- Wu Ch. Plasma free and protein-bound testosterone in hirsutism. Obstet.Gynecol 1982; 60:188-194.
- Abraham, G.E. (1969) Solid-phase radioimmunoassay of estradiol-17b./ clin. Endocr.Metab. 29, 866-870.