Thyroxine, Free (fT4) ELISA kit

The Free T4 ELISA is a competitive immunoassay. Competition occurs between Free T4 present in calibrators, controls, specimen samples and an enzyme-labelled antigen (HRP conjugate) for a limited number of anti- T4 antibody binding sites on the microplate wells. After a washing step that removes unbound materials, the TMB substrate (enzyme substrate) is added which reacts with HRP to form a blue-coloured product that is inversely proportional to the amount of Free T4 present. Following an incubation, the enzymatic reaction is terminated by the addition of the stopping solution, converting the colour from blue to yellow. The absorbance is measured on a microplate reader at 450 nm. A set of calibrators is used to plot a calibrator curve from which the amount of Free T4 in specimen samples and controls can be directly read.

The labelled T4 (HRP conjugate) employed in this assay system has shown no binding properties towards thyroxine-binding globulin (TBG) and human serum albumin (HSA). The binding sites on the microplates are designed to be of a low binding-capacity in order not to disturb the equilibrium between T4 and its carrying proteins. The assay is carried out under normal physiological conditions of pH, temperature and ionic strength.