The CanAg NSE EIA is a solid phase, noncompetitive immunoassay based on two monoclonal antibodies (derived from mice) directed against two separate antigenic determinants of the NSE molecule. The monoclonal antibodies (MAb) used bind to the gammasubunit of the enzyme and thereby detects both the gammagamma and the alphagamma form. Calibrators and samples are incubated together with biotinylated AntiNSE MAb E21 and horseradish peroxidase (HRP) labelled AntiNSE MAb E17 in streptavidin coated microstrips. After washing, buffered Substrate/Chromogen reagent (hydrogen peroxide and 3, 3’, 5, 5’ tetramethylbenzidine) is added to each well and the enzyme reaction is allowed to proceed. During the enzyme reaction a blue colour will develop if antigen is present. The intensity of the colour development is proportional to the amount of NSE present in the samples. The colour intensity is determined in a microplate spectrophotometer at 620 nm (or optionally at 405 nm after addition of Stop Solution). Calibration curves are constructed for each assay by plotting absorbance value versus the concentration for each calibrator. The NSE concentrations of samples are then read from the calibration curve.