Principle of the assay: 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 samples) and an enzymelabelled 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 free testosterone in the sample. A set of calibrators is used to plot a calibration curve from which the amount of free testosterone in samples and controls can be directly read. The Aviva free testosterone kit utilizes a highly specific rabbit antitestosterone polyclonal antibody at a low binding capacity (Keq x concentration) to keep minimum disturbances of the testosterone-protein equilibrium. The other components in the test system are also optimized in order to not alter the original free testosterone concentration.
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