Principle of the assay: 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 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 samples and controls can be directly read.
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