Description
Intended Uses: ACTH ELISA is intended for the quantitative determination of ACTH (Adrenocorticotropic Hormone) in human plasma.
Principle of the Assay: ACTH Immunoassay is a two-site ELISA [Enzyme-Linked ImmunoSorbent Assay] for the measurement of the biologically 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 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 patient samples are simultaneously incubated with t"~ enzyme labeled.:ntiiJody 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 and the enzyme bound to the solid phase is incubated with the substrate, tetramethylbenzidine (TMB). An acidic stopping solution is then added to stop the reaction and converts the color to yellow. The intensity of the yellow color is directly proportional to the concentration of ACTH in the sample. A dose response curve of absorbance unit vs. concentration is generated using results obtained from the calibrators. Concentrations of ACTH present in the controls and patient samples are determined directly from this curve