Adrenocorticotropic Hormone ELISA (ACTH ELISA)

The ACTH ELISA is a two-site enzyme-linked immunosorbent assay for the measurement of thebiologically active 39 amino acid chain of ACTH. A goat polyclonal antibody to human ACTH,purified by affinity chromatography, and a mouse monoclonal antibody to human ACTH arespecific for well-defined regions on the ACTH molecule. One antibody is prepared to bind onlythe C-terminal ACTH 34-39 and this antibody is biotinylated. The other antibody is prepared tobind only the mid-region and N-terminal ACTH 1-24 and this antibody is labeled with horseradishperoxidase [HRP] for detection.In this assay, calibrators, controls, or patient samples are simultaneously incubated with theenzyme 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 andthe enzyme bound to the solid phase is incubated with the substrate, tetramethylbenzidine (TMB).An acidic stop solution is then added to stop the reaction and converts the color to yellow. Theintensity of the yellow color is directly proportional to the concentration of ACTH in the sample.A dose-response curve of absorbance unit versus concentration is generated using resultsobtained from the calibrators. Concentrations of ACTH present in the controls and patient samplesare determined directly from this curve.