The CanAg CA15-3 EIA is a solid-phase, non-competitive immunoassay based upon the direct sandwich technique.
Calibrators, controls and patient samples are incubated together with biotinylated Anti-CA15-3 monoclonal antibody and horse-radish peroxidase (HRP) labelled Anti-CA15-3 monoclonal antibody in Streptavidin coated microstrips.
After washing, buffered Substrate/Chromogen reagent (hydro-gen peroxide and 3, 3’, 5, 5’ tetra-methylbenzidine) 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 is proportional to the amount of CA15-3 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 CA15-3 concentrations of patient samples are then read from the calibration curve.