Testerone ELISA Test Kit One Step Rapid TES Test Cassettes

Materials provided with Testosterone ELISA Test Kit:
1. Microplate: Antibody (goat anti-rabbit IgG) Coated Wells
2. Testosterone ELISA Reference Standard Set:6 sets
0, 0.1, 0.5, 2.0, 6.0 & 18.0 ng/ml in human serum
3. Rabbit Anti-Testosterone Reagent in BSA
4. Testosterone EIA HRP- Conjugate Reagent
5. Testosterone Control 1 and 2 in human serum
6. Testosterone ELISA TMB Reagent
7. Stop Solution, 1N HCl

Materials required, not provided:
1. Distilled or deionized water
2. Precision pipettes & tips
3. Vortex mixer or equivalent
4. EIA kit Microplate Washer
5. EIA kit Microplate Reader at 450 nm wavelength

Testosterone ELISA Kit Background Information
Testosterone (17 B-hydroxyandrost-4-one-3-one) is a C19 steroid with an unsaturated bond. 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 with 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.

Testosterone ELISA Test 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 10 ul of Testosterone standards, controls, patient samples, 100 ul Testosterone-HRP conjugate reagent and 50 ul rabbit anti-Testosterone reagent at 37C 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 450 nm. 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.

Testosterone ELISA Kit Precision & Recovery
1. Precision
a. Intra-Assay Precision
Within-run precision was determined by replicate determinations of
four different serum samples in one assay.
Refer to Testosterone EIA insert for Within-assay variability table.
b. Inter-Assay Precision
Between-run precision was determined by replicate measurements of
six different serum samples over a series of individually calibrated
assays. Refer to Testosterone EIA insert for Within-assay variability table.
2. Recovery Study
Various patient serum samples of known Testosterone levels were
combined and assayed in duplicate. The mean recovery was 95.3%.