The CanAg CA242 EIA is a solid-phase, non-competitive immunoassay based on the direct sandwich technique.
Calibrators, controls and patient samples are incubated together with biotinylated anti-CA242 monoclonal antibody (MAb) C241 in Streptavidin coated microstrips.
CA242 present in calibrators, controls or samples is absorbed to the Streptavidin coated microstrips by the biotinylated anti-CA242 MAb during the incubation.
The strips are then washed and incubated with horseradish peroxidase (HRP) labelled Anti-CA242 MAb C242. After washing, buffered Substrate/ Chromogen reagent (hydrogen 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 CA242 antigen 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 CA242 concentrations of patient samples are then read from the calibration curve.