This TNF-β enzyme linked immunosorbent assay (ELISA) applies a technique called a quantitative sandwich immunoassay. The microtiter plate provided in this kit has been pre-coated with a monoclonal antibody specific to TNF-β. Standards or samples are then added to the appropriate microtiter plate wells and incubated. After washing to remove unbound TNF-β and other components of the sample, biotin-conjugated polyclonal antibody specific to TNF-β is added and incubated. If present, TNF-β will bind and become immobilized by the antibody pre-coated on the wells and then become “sandwiched” by the biotin conjugate. In order to quantitatively determine the amount of TNF-β present in the sample, Avidin conjugated to Horseradish Peroxidase (HRP) is added to each microplate well and incubated. Avidin is a tetramer containing four identical subunits, each having a high affinity-binding site for biotin. The wells are thoroughly washed to remove all unbound HRP-conjugated Avidin and 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-β, biotin-conjugated antibody and enzyme-conjugated Avidin will exhibit a change in colour. The enzyme-substrate reaction is terminated by the addition of a sulphuric acid solution and the colour change is measured spectrophotometrically at a wavelength of 450 nm ± 2 nm.

This kit includes two calibration diluents (Calibrator Diluent I for serum/plasma testing and Calibrator Diluent II for cell culture supernatant testing) in order to measure the concentration of TNF-β in the samples. According to the testing system, the standard provided is diluted (2-fold) with the appropriate Calibrator Diluent and assayed at the same time as the samples. This allows the operator to produce a standard curve of Optical Density (O.D) versus TNF-β concentration (pg/mL). The concentration of TNF-β in the samples is then determined by comparing the O.D. of the samples to the standard curve.