Description:
An enzyme immunoassay for the quantitative determination of total thyroxine (T4) in serum.
The thyroid hormones thyroxine (T4) and triiodothyronine (T3), are synthesized and stored in the thyroid gland and circulate in the bloodstream mostly bound to the plasma protein, thyroxine binding globulin (TBG). The thyroid gland and associated hormones are a major component of the endocrine system. They exert powerful and essential regulatory influences on growth, differentiation, cellular metabolism, and general hormonal balance of the body. Proteolytic cleavage of follicular thyroglobulin releases T4 into the bloodstream. Greater than 99% of T4 is reversibly bound to three plasma proteins in blood - thyroxine binding globulin (TBG) binds 70%, thyroxine binding pre-albumin (TBPA) binds 20%, and albumin binds 10%. Approximately 0.03% of T4 is in the free, unbound state in blood at any one time. Diseases affecting thyroid function may present a wide array of confusing symptoms. Measurement of total T4, TSH, Free T3 and Free T4 by immunoassay are reliable and convenient methods to determine the presence of thyroid disorders in patients. Increased levels of T4 have been found in hyperthyroidism due to Grave’s disease and Plummer’s disease and in acute and subacute thyroiditis. Low levels of T4 have been associated with congenital hypothyroidism, myxedema, chronic thryoiditis (Hashimoto’s disease), and with some genetic abnormalities.
To measure T4 by competitive immunoassay techniques, a sample of serum containing the T4 to be quantified is mixed with labeled T4 and T4 antibody. The labeled T4 contains 8-anilino-1-napthalene sulfonic acid (ANS) to inhibit binding of T4 to serum proteins, which would otherwise interfere with the assay. During incubation, a fixed amount of labeled T4 competes with the unlabeled T4 in the sample, standard, or quality control serum for a fixed number of binding sites on the specific T4 antibody. Separation of the unbound T4 from antibody-bound T4 and the subsequent measurement of the labeled fraction of the bound phase completes the test. By comparing results of the unknown sample with those obtained from a series of T4 calibrators, an accurate measurement of the T4 concentration in the sample can be obtained. In the T4 ELISA (EIA-1781), antibody to T4 is coated on a solid phase (microtiter well). A measured amount of serum and a constant amount of T4 labeled with horseradish peroxidase are added. During incubation, T4 in the sample and enzyme-labeled T4 compete for the limited binding sites on the T4 antibody. After a 60-minute incubation at room temperature, the solid phase is washed with water to remove unbound-labeled T4. A solution of tetramethylbenzidine (TMB) is added and incubated for 20 minutes, resulting in the development of a blue color. The color development is stopped with the addition of 1N HCI, and the resulting yellow color 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 T4 in the sample. By reference to a series of calibrators processed in the same way, the concentration of T4 in the unknown sample is determined.