In order to quantitate the amount of CRP present in the sample, a standardized preparation of horseradish peroxidase (HRP)-conjugated antibody specific for CRP is added to each well to "sandwich" the CRP immobilized during the second incubation. The wells are thoroughly washed to remove all unbound HRP-conjugated antibodies and a TMB (3,3'5,5' tetramethyl-benzidine) substrate solution is added to each well. The enzyme (HRP) and substrate are allowed to react over a short incubation period. Only those wells that contain CRP and enzyme-conjugated antibody will exhibit a change in colour. The enzyme-substrate reaction is terminated by the addition of an acid solution and the colour change is measured spectrophotometrically at a wavelength of 450 nm ± 2 nm.

In order to measure the concentration of CRP in the samples, this kit standard (ready-to use) is assayed at the same time as the samples (diluted if necessary with Sample Diluent). This allows the operator to produce a standard curve of Optical Density (O.D.) versus CRP concentration (mg/L). The concentration of CRP in the samples is then determined by comparing the O.D. of the samples to the standard curve.