Principle of Assay
This human IL-22 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 human IL-22. Standards or samples are then added to the appropriate microtiter plate wells and incubated. Human IL-22, if present, will bind and become immobilized by the antibody pre-coated on the wells. Then, a preparation of biotin conjugated detection antibody for human IL-22 is added to each well and incubated. The Biotin conjugated antibody will bind to human IL-22 during incubation. The microtiter plate wells are thoroughly washed to remove unbound components in the samples and biotin conjugate preparation. Avidin has a very high affinity to biotin. In order to quantify the amount of human IL-22 present in the sample, a preparation of horseradish peroxidase (HRP)-conjugated Avidin is added to each well. HRPwill be linked to the IL-22 detection antibody through high affinity binding of Biotin and Avidin, and connect indirectly with human IL-22 immobilized on the well. After incubation, the wells are thoroughly washed to remove all unboundHRP-conjugated antibodies and aTMB(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 human IL-22 will exhibit a change in colour and the extent of colour change is proportional to the amount of human IL-22 in 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 wavelength of 450 nm ± 2 nm.
In order to measure the concentration of human IL-22 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 IL-22 concentration (pg/mL). The concentration of IL-22 in the samples is then determined by comparing the O.D. of the samples to the standard curve.
