This Thrombospondin 2 enzyme linked immunosorbent assay (ELISA) applies a technique called a quantitative sandwich immunoassay. The microtiter plate has been pre-coated with an Anti-Thrombospondin 2 Antibody. Standards or samples are then added to the microtire plate wells and Thrombospondin 2, if present, will bind to the antibody pre-coated on the wells. In order to quantify the amount of Thrombospondin 2 present in the sample, Anti-Thrombospondin 2 Antibody (Biotin) is added to each well to "sandwich" the Thrombospondin 2 immobilised on the plate. The microtiter plate then undergoes incubation, followed by thorough washing of the wells to remove all unbound components. Next, Avidin-Biotin-Peroxidase Complex is added to each well for a short incubation period, followed by thorough washing of the wells to remove all unbound conjugates. 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 Thrombospondin 2 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.