The Bovine Cardiac Tn-I ELISA Kit uses two different affinity purified antibodies. One is used for solid phase immobilization (on the microtiter wells). The second is conjugated to horseradish peroxidase (HRP). The serum sample is allowed to react simultaneously with the two antibodies, resulting in cTnI being sandwiched between the solid phase and HRP-conjugated antibodies. After incubation for one hour at room temperature on a plate shaker, the wells are washed to remove unbound HRP conjugated antibodies. A solution of tetramethylbenzidine (TMB), anHRP substrate, is then added and incubated for 20 minutes, resulting in the development of a blue color. The color development is stopped by addition of 1N HCl, changing the color to yellow. The concentration of cTnI is proportional to the absorbance at 450 nm.