This GM-CSF 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 GM-CSF. Standards or samples are then added to the appropriate microtiter plate wells and incubated. GM-CSF, 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 GM-CSF and other components of the sample. In order to quantitatively determine the amount of GM-CSF present in the sample, a standardized preparation of horseradish peroxidase HRP-conjugated monoclonal antibody specific for GM-CSF is added to each well to “sandwich” the GM-CSF 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 GM-CSF 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 450 nm ± 2 nm.

In order to measure the concentration of GM-CSF in the samples, this kit includes two diluents (Calibrator Diluent I for serum/plasma testing and Calibrator Diluent II for cell culture supernatant testing). According to the testing system, the provided standard is diluted (2-fold dilution series) 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 GM-CSF concentration (pg/mL). The concentration of GM-CSF in the samples is then determined by comparing the O.D. of the samples to the standard curve.