Enzyme Immunoassay for the Quantitative Determination of Total Thyroxine (T4) in Human Serum

for in vitro diagnostic use

Product Description
L-Thyroxine (T4) is a hormone that is synthesized and stored in the thyroid gland. 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 by immunoassay is the most reliable and convenient screening test available to determine the presence of thyroid disorders in patients. Increased levels of T4 have been found in hyper-thyroidism 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.

Principle
In the T4 EIA, a certain amount of anti-T4 antibody is coated on microtiter wells. A measured amount of patient serum, and a constant amount of T4 conjugated with horseradish peroxidase are added to the microtiter wells. During incubation, T4 and conjugated T4 compete for the limited binding sites on the anti-T4 antibody.
After a 60 minutes incubation at room temperature, the wells are washed 5 times by water to remove unbound T4 conjugate. A solution of TMB Reagent is then added and incubated for 20 minutes, resulting in the development of blue color.
The color development is stopped with the addition of Stop Solution, and the absorbance is measured spectrophotometrically at 450nm.
The intensity of the color formed is proportional to the amount of enzyme present and is inversely related to the amount of unlabeled T4 in the sample. By reference to a series of T4 standards assayed in the same way, the concentration of T4 in the unknown sample is quantified.

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