Principle of Assay
This mouse IL-6 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 for mouse IL-6. Standards or samples are then added to the appropriate microtiter plate wells and incubated. Mouse IL-6, if present, will bind and become immobilized by the antibody pre-coated on the wells. The microtiter plate wells are thoroughly washed to remove unbound mouse IL-6 and other components of the sample. In order to quantify the amount of mouse IL-6 present in the sample, a standardized preparation of biotin conjugated anti-mouse IL-6 detection antibody is added to each well. The detection antibody will bind to mouse IL-6 and be immobilized during the incubation. The plate is washed again and Avidin conjugated horseradish peroxidase (HRP) is added to each wells and incubated. Since Avidin has a very affinity to biotin, HRP will be linked indirectly to the detection antibody on the plate via the binding with of avidin to biotin. The microtiter plate is thoroughly washed to remove all unbound HRP 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 mouse IL-6 and 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.
In order to measure the concentration of mouse IL-6 in the samples, this kit contains two calibration diluents (Calibrator Diluent I for serum/plasma testing and Calibrator Diluent II for cell culture supernatant/ urine testing). According to the testing system, the provided standard 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 IL-6 concentration (pg/mL). The concentration of IL-6 in the samples is then determined by comparing the O.D. of the samples to the standard curve. This IL-6 ELISA is a 3 hour solid-p
