Human Free Thyroxine (Free T4) ELISA Kit

Thyroxine, the principal thyroid hormone, circulates in blood almost completely bound to carrier proteins. The main carrier is thyroxine-binding globulin (TBG). However, only the free (unbound) portion of thyroxine is responsible for the biological action. Further, the concentrations of the carrier proteins are altered in many clinical conditions, such as pregnancy. In normal thyroid functions as the concentrations of the carrier proteins alters, the total thyroxine level changes so that the free thyroxine concentration remains constant. Thus, measurements of free thyroxine concentration correlate better with clinical status than total thyroxine levels. For example, the increase in total thyroxine associated with pregnancy, oral contraceptives and estrogen therapy occassionally results in total T4 levels over the limits of normal while the free thyroxine concentration remains in the normal reference range. Masking of abnormal thyroid function can also occur in both hyper and hypothyroid conditions by alterations in the TBG concentration. The total T4 can be elevated or lowered by TBG changes such that the normal reference level result. Again, the free thyroxine concentration typically uncovers the patient’s actual clinical status. 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-T4 conjugate (analog method) is added, then the reactants are mixed. A competition reaction results between the enzyme conjugate and the free thyroxine for a limited number of antibody combining sites immobilized on the well. After the completion of the required incubation period, the antibody bound enzyme-thyroxine conjugate is separated from the unbound enzyme-thyroxine 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 free thyroxine concentration permits construction of a graph of activity and concentration. From comparison to the dose response curve, an unknown specimen’s activity can be correlated with free thyroxine concentration.