This assay employs the quantitative sandwich enzyme immunoassay technique. A monoclonal antibody specific for IL-9 has been pre-coated onto a microplate. Standards and samples are pipetted into the wells and IL-9 present is bound by the immobilized antibody. After washing away unbound substances, a biotin-linked antibody specific for IL-9 is added to the wells. Following a wash to remove unbound biotin labelled antibody, an avidin-HRP conjugate is added to each wells. Avidin binds to biotin labelled antibody with high affinity. After TMB substrate solution is added to the wells, colour will develop in proportion to the amount of IL-9 bound in the initial step. The colour development is stopped and the intensity of the colour is measured.

In order to measure the concentration of IL-9 in the samples this kit 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-9 concentration (pg/mL). The concentration of IL-9 in the samples is then determined by comparing the O.D. of the samples to the standard curve.