IVD Reagents

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The Eclectica TSH assay for the Eclectica analyzers has been designed for the quantitative determination of thyroid stimulating hormone (thyrotropin, TSH) in human serum and heparinized plasma. The method can be used without dilution for samples containing 0.05 to 50 µIU/ml of TSH. Primary calibration is against the WHO 2nd International Reference Preparation (80/558) for immunoassay of pituitary TSH (1).

Thyroid stimulating hormone (TSH) is a glycoprotein hormone produced by the anterior pituitary gland, with a molecular weight of approximately 28,000 daltons. TSH consists of two non-covalently associated subunits, alpha and beta (2). The alpha subunit is of 89 amino acids and is very similar in its structure to the alpha chains of luteinizing hormone (LH) and follicle stimulating hormone (FSH). The alpha chain of chorionic gonadotrophin is also closely related, although it contains 92 amino acids (3). The beta subunits confer the distinctive biological and immunological specificities on the whole molecules. Neither subunit is active on its own. The beta subunit controls the binding of the intact hormone to specific receptors on target cells; the alpha subunit then activates the cellular messenger, probably adenylatecyclase -cAMP (4).

Synthesis and release of TSH is stimulated by thyrotropin-releasing hormone (TRH). This hormone is a tripeptide, produced by the hypothalamus and conducted directly to the anterior pituitary by specialized vessels in the pituitary stalk (5). The stimulation of TSH release is subordinate to the negative feedback effects of the thyroid hormones thyroxine (T4) and triiodothyronine (T3) (6).

TSH acts on a specific target gland - the thyroid. Its main function is to regulate the synthesis and release by the thyroid of the iodothyronine hormones T4 and T3 (7). T4 and T3 exercise a general control on the level of metabolic activity on many other organ systems of the body (8); they also control the pituitary output of TSH through a negative feedback mechanism (9). The interlocking system of hormone actions may be perturbed by abnormalities in any of its components.

A relative excess of thyroid hormones causes hyperthyroidism, characterized by generally elevated metabolic activity. This hyperthyroidism may be primary or secondary, depending on the site of the malfunction (10). Much more common is primary hyperthyroidism, where all or part of the thyroid gland becomes overactive, no longer requiring stimulation by TSH for production and release of T4 and T3. Conditions such as Graves’ disease, adenomas, or autonomous nodules may cause overproduction of thyroid hormones, leading to elevated circulating levels of T4 and and T3. The pituitary responds to these elevations by reducing its output of TSH, so that TSH often becomes undetectable in serum. Rarely, a pituitary or hypothalamic tumor will result in excessive production of TSH. This overstimulates thyroid production of T4 and T3, causing secondary hyperthyroidism. The ability of immunometric assays to measure TSH in all categories of thyroid disease has lead to their increasing use as first-line tests of thyroid function in clinical practice (11,12).

Hypothyroidism may also be primary or, more rarely, secondary (10). Inability of the thyroid to produce sufficient T4 and T3 (primary hypothyroidism, from iodine deficiency goitre, for example) leads to a general slowing down of metabolic activities due to the low circulating levels of thyroid hormones. These same low levels of T4 and T3 release the pituitary from its feedback inhibition, and TSH production, and hence circulating levels, rise above normal. If the defect is in the pituitary (or hypothalamus), the consequent low levels of TSH will be insufficient to maintain adequate output of T4 and T3, resulting in secondary hypothyroidism.

This straightforward picture of the inter-relationships of TSH and the thyroid hormones covers the majority of cases of thyroid problems. However, variations of these relationships are seen in clinical practice. Most patients who become hyper - or hypothyroid do so gradually. Thus there may be intermediate stages in the development of the disease where one parameter is normal and another abnormal. For example, in a patient whose thyroid is slowly failing, normal levels of T4 and (especially) T3 may be maintained by increased production of TSH. This is sometimes called compensated hypothyroidism.

The Eclectica TSH assays for the Eclectica analyzers consist of:

1. Eclectica TSH Reagent Kit: specific reagents of Eclectica TSH kit to execute the test and to verify the standardization.
2. Eclectica TSH Calibration Kit: for standardization.
3. Eclectica Immunoassay Common Reagents Kit: for general chemistry reaction.
4. Eclectica Immunoassay Wash Solution: Wash Solution specific for Immunological reaction.
5. Eclectica Systemic Wash Solution: Wash Solution of Eclectica system.

In the Eclectica TSH assay, two high affinity monoclonal antibodies are used in an immunoenzymetric assay system which incorporates magnetic solid phase separation. All incubation times and reagent volumes are determined by an assay specific software protocol.

Fixed amounts of fluorescein-conjugated anti-TSH monoclonal antibody and anti-TSH monoclonal antibody conjugated to alkaline phosphatase are added to a sample, control, or standard. The reactants are incubated at 37°C. During the incubation fluorescein-anti-TSH monoclonal antibody binds to a discrete site on the TSH molecule. Alkaline phosphatase anti-TSH monoclonal antibody binds to a second site on the TSH molecule forming a sandwich.

At the end of the incubation period anti-fluorescein coupled to a magnetic solid phase is added in excess. This rapidly and specifically binds to the TSH monoclonal antibody complex and is sedimented in a magnetic field.

After aspirating the liquid phase and washing the solid phase, a solution of the enzyme substrate, phenolphthalein monophosphate is added to the incubation cell and the each cell incubated at 37°C. After incubation the enzyme reaction is stopped by the addition of a stop reagent and the intensity of the color developed is measured photometrically.

The intensity of the color developed 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 control is then determined by interpolation from a stored standard curve.

Eclectica TSH Reagent Kit

Eclectica TSH Calibration Kit

Eclectica Immunoassay Common Reagents Kit

Eclectica Immunoassay Wash Solution

Eclectica Systemic Wash Solution

1700006 Eclectica Cleaning Kit