This proBNP enzyme linked immunosorbent assay (ELISA) applies a technique called a quantitative sandwich immunoassay. The microtiter plate has been pre-coated with an Anti-proBNP Antibody. Standards or samples are then added to the microtire plate wells and proBNP, if present, will bind to the antibody pre-coated on the wells. In order to quantify the amount of proBNP present in the sample, Anti-proBNP Antibody (HRP) is added to each well to "sandwich" the proBNP immobilised on the plate. The microtiter plate then undergoes incubation, followed by thorough washing of the wells to remove all unbound components. Next, 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 proBNP will exhibit a change in colour. The enzyme-substrate reaction is terminated by the addition of a stop solution and the colour change is measured spectrophotometrically at a wavelength of 450nm ± 2nm.