This mouse IL-5 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-5. When standards or samples are added to the appropriate microtiter plate wells, mouse IL-5 in the standards or samples will be immobilized by the precoated antibody during incubation. Then, a biotin-conjugated antibody preparation specific for mouse IL-5 is added to each well and incubated. The biotin labelled antibody attaches to the wells by binding to mouse IL-5. After plate washing, other proteins, components and unattached biotin labelled antibody is removed. After that, avidin-horseradish peroxidase (HRP) conjugate is added to each well. Avidin has a very high affinity for biotin, thus, it links the tracer (HRP) sturdily to the biotin labelled antibody. The wells are thoroughly washed to remove all unbound avidin-HRP conjugate 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 wells that contain mouse IL-5 will exhibit a change in colour. The extent of colour change is proportional to the quantity of mouse IL-5 present in the standards/samples. The enzyme-substrate reaction is terminated by the addition of a sulphuric acid solution and the colour change is measured spectrophotometrically at a wave length of 450 nm ± 2 nm.

In order to measure the concentration of mouse IL-5 in the samples, this kit contains two calibration 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) 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-5 concentration (pg/mL). The concentration of mouse IL-5 in the samples is then determined by comparing the O.D. of the samples to the standard curve.