Rabbit Myoglobin ELISA is based on the principle of a solid phase enzyme-linked immunosorbent assay (ELISA). A monoclonal anti-myoglobin antibody is used for solid phase immobilization (on the microtiter wells) and polyclonal anti-myoglobin antibody conjugated to horseradish peroxidase is in the antibody-enzyme conjugate solution. The test sample is allowed to react simultaneously with the two antibodies, resulting in the myoglobin molecules being sandwiched between the solid phase and enzyme-linked antibodies. After one hour incubation at room temperature, the wells are washed to remove unbound labeled antibodies. A TMB (Tetramethyl-benzidine) 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 myoglobin is proportional to the color intensity of the test sample. Absorbance is measured spectrophotometrically at 450 nm.
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