MicroELISA

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The EIAgen U-TSH Kit has been designed for the quantitative determination of human Thyroid Stimulating Hormone (TSH) in human serum or plasma. The standards are calibrated against WHO 2^nd International Reference Preparation (IRP) for human TSH (80/558) (1).

Thyroid-stimulating hormone (TSH), or thyrotrophin, is a glycoprotein with a molecular weight of about 28,000 secreted by the pituitary gland. TSH is composed of two subunits of approximately equal size, called a and b. Other hormones such as luteinizing hormone (lH) and follicle-stimulating hormone (FSH), both secreted by the pituitary, and chorionic gonadotrophin (hCG), produced by the placenta, have a subunits virtually identical to that of TSH, but there are important differences in their b subunits (2). These differences confer biological specificity on the complete molecules and allow them to be distinguished in immunoassays.

Like the other glycoprotein hormones, TSH has a specific site of action which is the thyroid gland. Its main function is to regulate the release of thyroxine (T4) and the more biologically active triiodothyronine (T3), and to control different stages of their synthesis (4). These thyroid hormones circulate bound to thyroxine-binding globulin (TBG) and to a lesser extent, to albumin and pre-albumin. In their unbound form, they have widespread effects on other organ systems of the body, exerting a general control on the level of metabolic activity (6). They also exert negative feedback on the pituitary, inhibiting the release of TSH.

Synthesis and release of TSH is stimulated by thyrotrophin-releasing hormone (TRH). This hormone is a triceptide, produced by the hypothalamus and conducted directly to the anterior pituitary by specialized vessels in the pituitary stalk (7). The stimulation of release is subordinate to the negative feedback effect of the thyroid hormones (5).

In the normal individual, these hormones interact to maintain a dynamic equilibrium. TRH stimulates the pituitary to produce and release TSH which causes the thyroid gland to release T3 and T4. Circulating levels of T3 and T4 feed back to the pituitary, inhibiting the release of TSH (9). In this way, a metabolic equilibrium is preserved. This balance can be upset, however, by abnormalities in any stage of the negative-feedback cycle with clinical manifestations resulting from over-production (hyperthyroidism) or under-production (hypothyroidism) of T4 and/or T3.

Hyperthyroidism, caused by a relative excess of thyroid hormone, can be primary or secondary, depending on whether the abnormality is in the thyroid itself or in the pituitary. A variety of conditions (for example, Graves’ disease, adenoma, goiter, autonomous nodules) can cause all or part of the thyroid to become overactive, no longer requiring stimulation by TSH for release of T4 and T3. These hormones will feed back to the pituitary, inhibiting production of TSH. In patients with primary hyperthyroidism, high levels of T3/T4 will be found, along with low or zero levels of TSH.

Elevated levels of T3/T4 may also result from hyperstimulation of the thyroid by excessive TSH; this condition constitutes secondary hyperthyroidism, where all the hormones will be found at elevated levels.

The opposite condition, hypothyroidism, resulting from a relative lack of thyroid hormone, can also be primary or secondary. Inability of the thyroid to produce T3/T4 (primary hypothyroidism) - for example, from iodine deficiency - is characterized by low levels of T3/T4 and, because the feedback inhibition is removed, high levels of TSH. Reduced pituitary release of TSH causes secondary hypothyroidism by providing insufficient stimulus to the thyroid for T3/T4 production. In this case, all hormone levels are low. In hypothyroidism, diagnostic accuracy may be improved by performing a TRH stimulation test. Patients with primary hypothyroidism usually show an exaggerated response, while those with secondary hypothyroidism have a reduced response or none at all.

The measurement of TSH with EIAgen U-TSH can also be beneficial during therapy of primary thyroid disorders. As a result of its ability to measure subnormal TSH levels accurately, the EIAgen assay can be used during treatment to follow hyperthyroid as well as hypothyroid patients.

The EIAgen U-TSH assay is based on the one step immunoenzymatic sandwich principle, in conjunction with the Biotin-Streptavidin technology.

Two monoclonal anti-TSH of high affinity and specificity are used: one is labelled with Horse Radish Peroxidase (HRP) and the other with Biotin, while the microplate wells are coated with Streptavidin.

Samples, calibrators and controls are dispensed into the wells, followed by the mixture of the two labelled anti-TSH.

During the incubation the two monoclonals bind the TSH molecule to two different and specific sites, and contemporaneously, the Streptavidin immobilizes the forming immunological sandwich to the wells through the binding to the biotin moiety of the biotinilated antibody.

After washing to eliminate the not reacted species the mixture of chromogen/substrate is added.

The reaction is then blocked by adding the Stop Solution and the developed colour is measured photometrically.

The intensity of the colour is directly proportional, within the working range of the assay, to the concentration of TSH in the sample. The concentration of TSH in a patient sample or controls is then determined by interpolation on the calibration curve.

Each kit contains sufficient reagents to perform 96 tests (code LI4025K).

Bring reagents to room temperature before use.