The AffiELISA® Zebrafish TNFa Kit uses Sandwich enzyme immunoassay. The provided microtiter plate comes pre-coated with an antibody specific to Tumor Necrosis Factor Alpha (TNFa) . To perform the assay, standards or samples are added to designated wells on the microtiter plate. Subsequently, a biotin-conjugated antibody specific to Tumor Necrosis Factor Alpha (TNFa) is introduced. Avidin conjugated to Horseradish Peroxidase (HRP) is then added to each well, followed by an incubation period. After TMB substrate solution is added, only those wells that contain Tumor Necrosis Factor Alpha (TNFa) , biotin-conjugated antibody and enzyme-conjugated Avidin will exhibit a change in color. The enzyme-substrate reaction is terminated by the addition of sulphuric acid solution and the color change is measured spectrophotometrically at a wavelength of 450nm ± 10nm. By comparing the optical density (OD) of the samples to the standard curve, the concentration of Tumor Necrosis Factor Alpha (TNFa) in the samples can be determined.