A peroxidase-conjugated second antibody directed against the same antigen is given into the wells and after 20 minutes of incubation, the plate is washed again. A substrate solution is added and incubated for 20 minutes, resulting in the development of a blue color. The color development is inhibited by the addition of a Stop Solution, and the color turns yellow. The yellow color is measured photometrically at 450 nm. The concentration of the antigen is directly proportional to the color intensity of the test sample.