The AffiELISA® Rat pro interleukin 1 beta Kit uses Sandwich enzyme immunoassay. The provided microtiter plate comes pre-coated with an antibody specific to pro-IL1b (pro interleukin 1 beta) . To perform the assay, standards or samples are added to designated wells on the microtiter plate. Subsequently, a biotin-conjugated antibody specific to pro-IL1b (pro interleukin 1 beta) 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 pro-IL1b (pro interleukin 1 beta) , 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 pro-IL1b (pro interleukin 1 beta) in the samples can be determined.