The CanAg NSE EIA test is in solid phase. This non-competitive immunoassay is based on two monoclonal antibodies (from mice) directed against two separate antigenic determinants of the NSE molecule. The monoclonal antibodies (Mab) used relate to the  subunit of the enzyme, and therefore detect both ϒϒ  and α ϒ forms.

Calibrators and patient samples are incubated together, with biotinylated Anti-NSE Mab E21 and raIFO/rt peroxidase (HRP) labelled Anti-NSE Mab E17, in Streptavidin-coated microtiter strips.

After washing, the substrate/chromogen buffer reagent (hydrogen peroxide and 3, 3', 5, 5' tetra-methyl-benzidine) is added to each well and the enzymatic reaction can begin.

During the enzymatic reaction, a blue color develops if antigen is present. The intensity of color development is proportional to the amount of NSE present in the samples. Color intensity is determined by a microtiter plate spectrophotometer at 620 nm (or optionally at 405 nm) after addition of the Stop Solution.

Calibration curves are constructed for each test by plotting absorbance values against the concentration of each Calibrator. The NSE concentrations of the patient samples are then read from the calibration curve.

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