Principle of Assay
This assay applies the quantitative sandwich enzyme immunoassay technique. A monoclonal antibody specific to IL-13 has been pre-coated onto a microplate. Standards and samples are then added to the appropriate plate wells with a biotin-conjugated antibody preparation specific for IL-13 and incubated. IL-13 if present, will bind and become immobilized by the antibody pre-coated on the wells and then be "sandwiched" by biotin conjugate. Avidin is a tetramer containing four identical subunits that each has a high affinity-binding site for biotin. After washing away any unbound substances, avidin-Horseradish Peroxidase (HRP) will be added to each well and incubated. Following a wash to remove any unbound antibody-enzyme reagent, a substrate solution is added to the wells and only those wells that contain IL-13, biotin conjugated antibody and enzyme-conjugated Avidin will exhibit a change in colour. The colour development is stopped and the intensity of the colour is measured.
In order to measure the concentration of IL-13 in the samples, this package of reagent includes 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-13 concentration. The concentration of IL-13 in the samples is then determined by comparing the O.D. of the samples to the standard curve.
