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The 17α-OHP ELISA is a competitive immunoassay. Competition occurs between 17α-OHP present in calibrators, controls, specimen samples and an enzyme-labelled antigen (HRP conjugate) for a limited number of anti-17α-OHP antibody binding sites on the microplate wells. After a washing step that removes unbound materials, the TMB substrate (enzyme substrate) is added which reacts with HRP to form a blue-coloured product that is inversely proportional to the amount of 17α-OHP present. Following an incubation, the enzymatic reaction is terminated by the addition of the stopping solution, converting the colour from blue to yellow. The absorbance is measured on a microplate reader at 450 nm. A set of calibrators is used to plot a calibrator curve from which the amount of 17α-OHP in specimen samples and controls can be directly read.