The CEA ELISA test is based on the principle of a solid phase enzymelinked immunosorbent assay. The assay system utilizes a monoclonal antibody directed against a distinct antigenic determinant on the intact CEAmolecule is used for solid phase immobilization (on the microtiter wells). A goat anti-CEA antibody conjugated to horseradish peroxidase (HRP) is in the antibody-enzyme conjugate solution. The test sample is allowed to react simultaneously with the two antibodies, resulting in the CEA molecules being sandwiched between the solid phase and enzyme-linked antibodies. After a 1 hour incubation at room temperature, the wells are washed with water to remove unbound labeled antibodies. A solution of TMB Reagent is added and incubated 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 CEA is directly proportional to the color intensity of the test sample. Absorbance is measured spectrophotometrically at 450 nm.
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