Introduction and Explanation of the Test
Quantitative Analysis of Total Tyroxine (T4) concentration in human serum
Thyroid gland is the source of L- thyroxine (T4), Where (T4) is synthesized and stored. Thyroxine is one type of hormone, which circulates in the blood as an equilibrrium mixture of free and serum bound hormone. More than 99% of T4 is revesibly bound to three plasma proteins in blood – thyroxine binding globulin(TBG) 70%, albumin (10%) and thyroxine binding pre-albumin(TBPA) (20 %). Less than 0.03% T4 is present in the circulation as unbound state in the blood. This small T4 amount represents the physiologically available hormone which is biologically active.
The presence of thyoid disorders in patients is determined by Immunoassay test. This test is the most reliable convenient and fast screening test for the determination of total T4. Levels of T4 are found to increase in hyper-thyroidism due to Graves disease and Plummers disease and in acute and subacute thyroiditis. Low levels of T4 have been linked with congenital hypothyroidism, myxedema, chronic thyroiditis (Hashimotos disease), and with some genetic abnormalities.
Principle of the Test
In this T4 EIA, Some amount of anti-T4 antibody is coated on microtiter wells. A constant amount of T4 conjugated with Alkaline phosphatase and a measured amount of human serum are added to the micro titer wells and sake before incubation. During incubation , T4 and conjugated T4 compete for the limited binding sites on the anti T4 antibody. After 30 minutes incubation at 370C temperature, the wells are washed by Tris buffer 4 times to removed unbound T4 conjugate. A constant amount of chemiluminescent Reagent is then added in each well and sake well and keep the plate in the sample chamber of the luminometer and measure the intensity of l8ght (in RLU) after every 15 minutes at 450 nm. The intensity of the chemiluminescent light is proportional to the amount of enzyme present and is inversely proportional to the amount of unlabeled T4 in the sample. T4 standards is tested in the same way as reference, and then calculate the T4 concentration in the unknown sample.