Calcitonin ELISA

The Calcitonin Immunoassay is a two-site ELISA for the measurement of the biologically intact32 amino acid chain of Calcitonin. It utilizes two different mouse monoclonal antibodies to humancalcitonin specific for well-defined regions on the calcitonin molecule. One antibody binds only toCalcitonin 11-23 and this antibody is biotinylated. The other antibody binds only to Calcitonin 21-32 and this antibody is labeled with horseradish peroxidase [HRP] for detection.  In this assay, calibrators, controls, or patient samples are simultaneously incubated with the enzyme-labeled antibody and a biotin-coupled antibody in a streptavidin-coated microplate well. Thus, thecalcitonin in the sample is “sandwiched” between these two antibodies. At the end of the assayincubation, 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 stop solution isthen added to stop the reaction and converts the color to yellow. The color intensity is directlyproportional to the concentration of calcitonin in the sample. A dose response curve of absorbancevs. concentration is generated using results obtained from the calibrators. Concentration ofcalcitonin present in the controls and samples are determined directly from this curve.