Enzyme immunoassay for quantitative determination of thyroid-stimulating hormone (TSH) in human serum
TSH (Thyroid-stimulating hormone) is a glycoprotein hormone of approximate molecular weight of 28,000 daltons, secreted by the anterior pituitary gland. It consists of two non-covalently bound subunits, known as α and β. The α subunits are very similar in structure, but the β subunits differ markedly in amino acid sequence, and are responsible for the differences in the biological specificity of TSH. The main function of TSH is to develop the thyroid gland and also to stimulate and control the secretion of thyroxine (T4) and triiodothyronine (T3). Increase in serum concentrations of TSH is an early and sensitive indicator of decrease thyroid reserve and in conjunction with decreased T4 concentrations is diagnostic of primary hypothyroidism. The secretion and the liberation of TSH are stimulated by the hypothalamic tripeptide TRH (TSH Releasing Hormone) and are controlled by serum concentrations of the thyroid hormones: T4 and T3 through a negative feedback mechanism: in the thyreotrope cells, T4 is desiodinated in T3 which directly inhibits TSH secretion.
The DIAKEY TSH EIA is a solid-phase, non-competitive immunoassay based upon the sandwich technique. Two different monoclonal TSH antibodies are used. One antibody is coated on solid phase coated plate, the other, specific for the TSH and labeled with HRPO, is used as a conjugate. The conjugate antibody and the coated antibody react simultaneously with the TSH antigen present in the standards, controls and samples. Unbounded material is removed by a washing step. After washing, substrate reagent is added to each well and the enzyme reaction is allowed to proceed. During the enzyme reaction a blue color will develop if antigen is present. The intensity of the color is proportional to the amount of TSH present in the samples. The color intensity is determined in a microplate spectrophotometer at 450nm (reference 620nm).