Pregnenolone, ELISA, 96 tests
The principle of the following enzyme immunoassay test follows a two-step competitive binding scenario.
During the first incubation, competition occurs between an unlabeled antigen (present in calibrators, control and patient samples) and a biotin-labelled antigen (biotin conjugate) for a limited number of antibody binding sites on the microwell plate.
The washing and decanting procedures remove unbound materials. During the second incubation, the streptavidin-HRP (HRP conjugate) is added and binds to the biotin-conjugate. 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 pregnenolone in the sample. A set of calibrators is used to plot a calibration curve from which the amount of pregnenolone in patient samples and controls can be directly read.