This assay employs the competitive inhibition enzyme immunoassay technique. An antibody specific for Adrenocorticotropic Hormone (ACTH) has  been  pre-coated  onto  a  microplate.  A  competitive  inhibition  reaction  is  launched  between Horseradish Peroxidase (HRP) labeled Adrenocorticotropic Hormone (ACTH) and unlabeled Adrenocorticotropic Hormone (ACTH) (Standards or samples) with the pre-coated antibody specific for Adrenocorticotropic Hormone (ACTH). After incubation the unbound conjugate is washed off. The amount of bound HRP conjugate is reverse proportional to the concentration of  Adrenocorticotropic Hormone (ACTH) in the sample. After addition of the substrate solution, the intensity of color developed is reverse proportional to the concentration of  Adrenocorticotropic Hormone (ACTH) in the sample.