An enzyme immunoassay for the quantitative determination of caridac specific troponin I in serum.
The assay system utilizes four unique monoclonal antibodies directed against distinct antigenic determinants on the molecule. Three mouse monoclonal anti-troponin I antibodies are used for solid phase immobilization (on the microtiter wells). The fourth antibody is in the antibody-enzyme (horseradish peroxidase) conjugate solution. The test sample is allowed to react simultaneously with the four antibodies, resulting in the troponin I molecules being sandwiched between the solid phase and enzyme-linked antibodies. After a 90-minute incubation at room temperature, the wells are washed with water to remove unbound-labeled antibodies. A solution of tetramethylbenzidine (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 1N hydrochloric acid (HCl) changing the color to yellow. The concentration of troponin I is directly proportional to the color intensity of the test sample. Absorbance is measured spectrophotometrically at 450 nm.