The PSA ELISA test is based on the principle of a solid phase enzymelinked immunosorbent assay. The assay system utilizes a rabbit anti-PSA antibody directed against intact PSA for solid phase immobilization (on the microtiter wells). A monoclonal anti-PSA antibody conjugated to horseradish peroxidase (HRP) is in the antibody-enzyme conjugate solution. The test sample is allowed to react first with the immobilized rabbit antibody at room temperature for 60 minutes. The wells are washed to remove any unbound antigen. The monoclonal anti-PSA-HRP conjugate is then reacted with the immobilized antigen for 60 minutes at room temperature resulting in the PSA molecules being sandwiched between the solid phase and enzymelinked antibodies. The wells are washed with water to remove unboundlabeled antibodies. A solution of TMB Reagent is added and incubated at room temperature for 20 minutes, resulting in the development of a blue color. The color development is stopped with the addition of Stop Solution changing the color to yellow. The concentration of PSA is directly proportional to the color intensity of the test sample. Absorbance is measured spectrophotometrically at 450 nm.
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