The principle of the following enzyme immunoassay test follows the typical competitive binding scenario.

Competition occurs between an unlabeled antigen (present in calibrators, control and patient samples) and an enzyme-labelled antigen (conjugate) for a limited number of antibody binding sites on the microwell plate.

The washing and decanting procedures remove unbound materials. After the washing step, the enzyme substrate is added. The enzymatic reaction is terminated by addition of the stopping solution.

The absorbance is measured on a microtiter plate reader. The intensity of the colour formed is inversely proportional to the concentration of fT3 in the sample.

A set of calibrators is used to plot a calibration curve from which the amount of fT3 in patient samples and controls can be directly read. The labelled T3 (conjugate) employed in this assay system has shown no substantial binding properties towards thyroxine-binding globulin (TBG) or human serum albumin (HSA).

The binding sites on the microwell plates are designed to be of a low binding-capacity in order not to disturb the equilibrium between T3 and its carrying proteins. The assay is carried out under normal physiological conditions of pH, temperature and ionic strength.

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