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immunoassay. The assay makes use of two highly specific monoclonal antibodies: A monoclonal antibody specific for free PSA is immobilized onto the microplate and another monoclonal antibody specific for a different epitope of free PSA is conjugated to horse radish peroxidase (HRP conjugate).
In the first incubation step, free PSA present in the specimen samples, calibrators and controls is simultaneously bound by the immobilized antibody and the HRP conjugate antibody, thus forming a sandwich complex. Excess and unbound materials are removed by a washing step.
Next, the TMB substrate (enzyme substrate) is added which reacts with HRP to form a blue coloured product that is directly proportional to the amount of free PSA present. 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 free PSA in specimen samples and controls can be directly read.