This IFN-γ 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 specific for IFN-γ. Standards or samples are then added to the appropriate microtiter plate wells and incubated. IFN-γ, 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 IFN-γ and other components of sample. In order to quantitatively determine the amount of IFN-γ present in the sample, a standardized preparation of horseradish peroxidase (HRP)-conjugated monoclonal antibody specific for IFN-γ is added to each well to "sandwich" the IFN-γ immobilized during the first incubation. The microtiter plate then undergoes a second incubation. The wells are thoroughly washed to remove all unbound HRP-conjugated antibodies 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 IFN-γ and enzyme-conjugated antibody 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 IFN-γ 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 IFN-γ concentration (pg/mL). The concentration of IFN-γ in the samples is then determined by comparing the O.D. of the samples to the standard curve.