This ELISA kit uses the Competitive-ELISA principle. The micro ELISA plate provided in this kit has been pre-coated with ACTH. During the reaction, ACTH in the sample or standard competes with a fixed amount of ACTH on the solid phase supporter for sites on the Biotinylated Detection Ab specific to ACTH. Excess conjugate and unbound sample or standard are washed away, and Avidin-Horseradish Peroxidase (HRP) conjugate are added to each micro plate well and incubated. Then a TMB substrate solution is added to each well. The enzyme-substrate reaction is terminated by the addition of stop solution and the color turns from blue to yellow. The optical density (OD) is measured spectrophotometrically at a wavelength of 450 nm ± 2 nm. The concentration of ACTH in tested samples can be calculated by comparing the OD of the samples to the standard curve.