Principle of Assay
This mouse IFN-? enzyme-linked immunosorbent assay (ELISA) applies a technique called a quantitative sandwich immunoassay. The microtiter plate provided in this kit has been precoated with a monoclonal specific for mouse IFN-?. Standards or samples are then added to the appropriate microtiter plate wells and incubated. In order to quantitatively determine the amount of mouse IFN-? present in the sample, a biotin-conjugated antibody specific for mouse IFN-? is added to each well to "sandwich" the IFN-? immobilized during the first incubation. The microtiter plate then undergoes a second incubation and the wells are thoroughly washed to remove all unbound Biotin-conjugated antibodies. Avidin can bind to biotin with high affinity and amplify the biotin signal. A preparation of avidin conjugated HRP is added to each well and incubated. The avidin HRP conjugate will bind to wells containing biotin. After washing the wells, the unbounded avidin-HRP conjugate is removed. 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 IFN-? and biotin-conjugated antibody, avidin conjugated HRP 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 450nm ± 2nm.
In order to measure the concentration of mouse IFN-? in the samples this kit contains two calibration diluents. (Calibrator Diluent I for serum/plasma testing and Calibrator Diluent II for cell culture supernatant). According to the testing system, the provided standard is 2 x fold serial diluted 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 mouse IFN-? concentration (pg/mL). The concentration of mouse IFN-? in the samples is then determined by comparing the O.D. of the samples to the standard curve.
