Chemiluminescence Immunoassay (CLIA) detection using microplate luminometers provides a sensitive, high throughput, and economical alternative to conventional colorimetric methodologies, such as Enzyme-linked immunosorbent assays (ELISA). ELISA employs a label enzyme and a colorimetric substrate to produce an amplified signal for antigen, haptens or antibody quantitation. This technique has been well established and considered as the technology of choice for a wide variety of applications in diagnostics, research, food testing, process quality assurance and quality control, and environmental testing. The most commonly used ELISA is based on colorimetric reactions of chromogenic substrates, (such as TMB) and label enzymes. Recently, a chemiluminescent immunoassay has been shown to be more sensitive than the conventional colorimetric method(s), and does not require long incubations or the addition of stopping reagents, as is the case in some colorimetric assays. Among various enzyme assays that employ lightemitting reactions, one of the most successful assays is the enhanced chemiluminescent immunoassay involving a horseradish peroxidase (HRP) labeled antibody or antigen and a mixture of chemiluminescent substrate, hydrogen peroxide, and enhancers. The CLIA Kits are designed to detect glow-based chemiluminescent reactions. The kits provide a broader dynamic assay range, superior low-end sensitivity, and a faster protocol than the conventional colorimetric methods. The series of the kits covers Thyroid panals, such as T3, T4, TSH, Hormone panels, such as hCG, LH, FSH, and other panals. They can be used to replace conventional colorimetric ELISA that have been widely used in many research and diagnostic applications. Furthermore, with the methodological advantages, Chemiluminescent immunoassay will play an important part in the Diagnostic and Research areas that ELISAs cannot do. The CLIA Kits have been validated on the MPL and MPL2 microplate luminometers from Berthold Detection System, Lus2 microplate luminometer from Anthos, Centro LB960 microplate luminometer from Berthold Technologies, and Platelumino from Stratec Biomedical Systems AG. We got
acceptable results with all of those luminometers.
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.
The Testosterone Chemiluminescence Immunoassay 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 μl of
Testosterone standards, controls, patient samples, 100 μl Testosterone-HRP conjugate reagent and 50
μl 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 chemiluminescent substrate is then added and read relative light units (RLU) with a Luminometer. The intensity of the emitting light is proportional to the amount of enzyme present and is inversely related to the amount of unlabeled TESTOSTERONE in the sample. By reference to a series of TESTOSTERONE standards assayed in the same way, the concentration of TESTOSTERONE in the unknown sample is quantified.