Description

The Diagnostic Automation, Inc. T uptake ELISA kit is intended for the measurement of the Total Amount of Binding Sites Available for the Thyroid Hormones in Human Serum or Plasma by a Microplate Enzyme Immunoassay thyroid gland under the regulatory control of thyrotropin hormone secretes thyroxine (T4) and triiodothyronine (T3) into the general circulation. The released hormones do not circulate as free molecules but are almost entirely (99.9%) bound to specific serum proteins. Three protein fractions with varying affinities and capacities for interaction with T3 and T4 have been identified by reverse flow paper electrophoresis. Thyroxine binding globulin (TBG) carries 65~75% of the total circulating concentration. Thyroxine binding pre-albumin (TBPA) has an intermediate avidity for thyroxine (carries approx.15~25%) but little if any avidity for triiodothyronine. Albumin with a low affinity but high capacity carries 10% of thyroxine and 30% of the available triiodothyronine. Since the metabolic processes are regulated entirely by the concentration of the free thyroid hormones, which are inversely related to the levels of the binding proteins, an assessment of the binding capacity of human serum was developed in 1957 by Hamolsky.

In this early method, radioactive T3 was added to a specimen of whole blood. After an incubation period, the mixture was centrifuged and the red cells washed. The radioactivity uptake of the red cells was inversely related to the binding capacity of the serum. Although this method had severe limitations, it proved to be a valuable diagnostic tool. Further technical improvements in the assay methodology of the T3-uptake test resulted as various separation agents such as coated charcoal, ion-exchange resins, denatured albumin, silicates, antibodies and organic polymers were employed in place of the red cells. This microplate enzyme immunoassay methodology provides the technician with optimum sensitivity while requiring few technical manipulations. In this method, serum reference, patient specimen, or control is first added to a microplate well. Enzyme-T3 conjugate and thyroxine (T4) are added, and then the reactants are mixed. The endogenous binding proteins of the sample react with the thyroxine, but not with the enzyme conjugate. This leads to a higher binding of the enzyme conjugate to the antibody combining sites, reactive for triiodothyronine and thyroxine, immobilized on the well as the binding capacity of the specimen increases.


After the completion of the required incubation period, the antibody bound enzyme- triiodothyronine conjugate is separated from the unbound enzyme- triiodothyronine conjugate by aspiration or decantation. The activity of the enzyme present on the surface of the well is quantitated by reaction with a suitable substrate to produce color. The employment of several serum references of known unsaturated thyroid hormone binding capacity permits construction of a graph of absorbance and concentration. From comparison to the dose response curve, an unknown specimen\\\\\\\'s absorbance can be correlated with thyroid hormone binding capacity.