Troponin ELISA - Troponin I

Enzyme Immunoassay for the Quantitative Determination of Cardiac-Specific Troponin-I in Human Serum

FOR RESEARCH USE ONLY. NOT FOR USE IN DIAGNOSTIC PROCEDURES

SUMMARY
Troponin is a complex of three regulatory proteins (troponin C, troponin I, and troponin T) that is integral to muscle contraction in skeletal muscle and cardiac muscle, but not smooth muscle. Discussions of troponin often pertain to its functional characteristics and/or to its usefulness as a diagnostic marker or therapeutic target for various heart disorders in particular as a highly specific marker for myocardial infarction or heart muscle cell death.

An increased level of the cardiac protein isoform of troponin circulating in the blood has been shown to be a biomarker of heart disorders, the most important of which is myocardial infarction. Raised troponin levels indicate cardiac muscle cell death as the enzyme is released into the blood upon injury to the heart.

PRINCIPLE OF THE TEST
The cTnI ELISA test is based on the principle of a solid phase enzyme-linked immunosorbent assay. 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.

EXAMPLE OF STANDARD CURVE
Results of a typical standard run with optical density readings at 450 nm shown in the Y axis against Troponin concentrations shown in the X axis. This standard curve is for the purpose of illustration only, and should not be used to calculate unknowns. Each user should obtain his or her own data and standard curve.