The ELISA is based on the principle of a solid phase enzyme-linked immunosorbent assay. The assay utilizes a mouse anti-human cytokine antibody for immobilization on the microtiter wells and goat anti-human cytokine antibodies along with streptavidin conjugated to horseradish peroxidase (HRP) for detection. The test sample is allowed to react simultaneously with the two antibodies, resulting in the molecules of the cytokine being sandwiched between the solid phase and enzyme-linked antibodies. After incubation, the wells are washed to remove unbound-labeled antibodies. A HRP substrate, TMB, is added to result in the development of a blue color. The color development is then stopped with the addition of Stop Solution changing the color to yellow. The concentration of the cytokine is directly proportional to the color intensity of the test sample. Absorbance is measured spectrophotometrically at 450 nm.