Principle of Assay
This IL-4 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 polyclonal antibody specific to IL-4. Standards or samples are then added to the appropriate microtiter plate wells with a biotin-conjugated monoclonal antibody preparation specific for IL-4 and incubated. IL-4 if present, will bind and become immobilized by the antibody pre-coated on the wells and then be “sandwiched” by biotin conjugate. The microtiter plate wells are thoroughly washed to remove unbound IL-4 and other components of the sample. In order to quantitatively determine the amount of IL-4 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 that each have 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 IL-4, biotin-conjugated antibody, and enzyme-conjugated Avidin will exhibit a change in colour. The enzymesubstrate 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.
