This TNF alpha enzyme linked immunosorbent assay (ELISA) applies a technique called a quantitative sandwich immunoassay. The microtiter plate has been pre-coated with an Anti-TNF alpha Antibody. Standards or samples are then added to the microtire plate wells and TNF alpha, if present, will bind to the antibody pre-coated on the wells. In order to quantify the amount of TNF alpha present in the sample, Anti-TNF alpha Antibody (Biotin) is added to each well to "sandwich" the TNF alpha 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 TNF alpha 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.