Principle of Assay
This CEA enzyme linked immunosorbent assay (ELISA) kit uses a technique called quantitative sandwich immunoassay. The microtiter plate provided in this kit has been pre-coated with a monoclonal antibody specific for CEA. During the assay, standards or samples are added to the microtiter plate wells and CEA, if present, will bind to the CEA antibody pre-coated on the wells. Then, a preparation of horseradish peroxidase (HRP)-conjugated CEA antibody is added to each well. The conjugated antibody will bind to the CEA immobilized on the plate after incubation. All unbound components will be removed by subsequent washing procedure. Afterwards, 3, 3', 5, 5' tetramethyl-benzidine(TMB), a substrate for HRP is added to each well. The colorimetric reaction is proportional to the CEA content in each well. The reaction is terminated by the addition of a sulphuric acid solution and the colour change is measured spectrophotometrically at the wavelength of 450nm ± 2nm.
In order to quantify the amount of CEA present in the sample, the calibration standards should be assayed at the same time as the samples to allow the operator to produce a standard curve of Optical Density (O.D.) versus CEA concentration (ng/mL). The concentration of CEA in the samples is then determined by comparing the O.D. of the samples to the standard curve.
