Total Thyroxine (T4) ENZYME IMMUNOASSAY TEST KIT (96 Wells)

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

Principle of the assay: To measure T4 by competitive immunoassay techniques, a sample of serum or plasma containing the T4 to be quantified is mixed with labeled T4 and T4 antibody.6,7 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 Aviva T4 EIA, 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.