Conventional elisa kit

Plasma samples were collected into heparinized tubes (Sarstedt, Nümbrecht, Germany) from the femoral vein at the 60th minute of reperfusion, and plasma was separated to determine cardiac troponin I (TnI) release after acute myocardial infarction. Plasma TnI concentration was determined by a conventional ELISA kit (Life Diagnostics, Inc, West Chester, PA) according to the recommendations of the manufacturer. Briefly, plasma samples were diluted 4 to 40 times according to the treatment protocol (ie, sham or ischemic) and to previous preanalyses to get absorbances in the range of standard absorbances. Diluted samples were allowed to react simultaneously with 2 antibodies against rat TnI (1 is immobilized on the microtiter wells, and the other is conjugated to horseradish peroxidase [HRP] in soluble phase), resulting in TnI being sandwiched between the solid phase and HRP‐conjugated antibodies. After 1 hour of incubation at room temperature on a plate shaker, the wells were washed with wash solution to remove unbound HRP‐conjugated antibodies. A solution of tetramethylbenzidine, a HRP substrate, was then added and incubated for 20 minutes, resulting in the development of a blue color. The color development was stopped by addition of 1N HCl, which changed the color to yellow. The concentration of TnI was proportional to the absorbance at 450 nm.