PRINCIPLE OF THE TEST The CBIACTH Immunoassay is a two-site lumELISA (Chemiluninescence Enzyme-Linked ImmunoSorbent Assay) for the measurement of the biologically active 39 amino acid chain of ACTH. A goat polyclonal antibody to ACTH, purified by affinity chromatography, and a mouse monoclonal antibody to ACTH are specific for well-defined regions on the ACTH molecule. One antibody is prepared to bind only the C-terminal ACTH 34-39 and this antibody is biotinylated. The other antibody is prepared to bind only the mid-region and N-terminal ACTH 1-24 and this antibody is labeled with horseradish peroxidase [HRP] for detection. In this assay, calibrators, controls, or samples are simultaneously incubated with the enzyme labeled antibody and a biotin coupled antibody in a streptavidin-coated microplate well. At the end of the assay incubation, the microwell is washed to remove unbound components. Upon the addition of the luminol substrate, the enzyme activity in the enzyme-bound fraction is directly proportional to the concentration of the ACTH in the sample. A standard curve is prepared relating light unit (RLU) to the concentration of the ACTH. Concentrations of ACTH present in the controls and samples are determined directly from this curve.