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 cortisol in the sample.
A set of calibrators is used to plot a calibration curve from which the amount of cortisol in patient samples and controls can be directly read.