Mouse IL-23 ELISA Assay
The Mouse IL-23 ELISA Assay is For Research Use Only
Size: 1×96 wells
Sensitivity: 7 pg/mL
Dynamic Range: 15.625 – 500 pg/ml
Incubation Time: 3 hours
Sample Type: Serum, Plasma, Cell Culture
Sample Size: 100 µl
Alternative Names: Interleukin 23
Interleukin 23 (IL-23) is a heterodimeric cytokine that is related to IL-12. It is composed of two disulfide-linked subunits, a 19 kDa (p19) subunit that is unique to IL-23, and a 40 kDa (p40, IL-12) subunit that is shared with IL-12. Mature mouse p19 and p40 share 88% and 92% aa sequence identity, respectively, with the corresponding rat subunits. IL-23 is produced by activated macrophages, microglia, and monocyte-derived dendritic cells in response to pathogens including certain bacteria and viruses and/or their components. The functional IL-23 receptor complex consists of two receptor subunits, the IL-12 receptor-1 subunit (IL-12 Rβ1) and the IL-23-specific receptor subunit (IL-23 R). IL-23 and IL-12 have overlapping and distinct biological activities. The IL-23 immune pathway induces the earliest recruitment of neutrophils to the site of infection, while the more classic host defense and cytotoxic response is stimulated by IL-12. IL-23 has a role in the development and maintenance of a T cell subset, designated Th17, that is characterized by the production of IL-17A, IL-17F, IL-6, and TNF-α. The IL-23/IL-17 axis is an important mediator of inflammation. In mouse models, transgenic over-expression of IL-23 leads to a lethal systemic inflammatory response. IL-23 effects on Th17 cells may also enhance the development of several models of autoimmune disease including experimental allergic encephalomyelitis (EAE), collagen-induced arthritis (CIA), colitis, and diabetes. IL-23 may also play a role in increased tumor growth associated with chronic inflammation.
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The Eagle Biosciences Mouse Interleukin 23 (IL-23) ELISA Assay Kit employs the quantitative sandwich enzyme immunoassay technique. A monoclonal antibody specific for IL-23 has been pre-coated onto a microplate. Standards and samples are pipetted into the wells and any IL-23 present is bound by the immobilized antibody. Following incubation unbound samples are removed during a wash step, and then a detection antibody specific for IL-23 is added to the wells and binds to the combination of capture antibody-IL-23 in sample. Following a wash to remove any unbound combination, and enzyme conjugate is added to the wells. Following incubation and wash steps a substrate is added. A colored product is formed in proportion to the amount of IL-23 present in the sample. The reaction is terminated by addition of acid and absorbance is measured at 450nm. A standard curve is prepared from seven IL-23 standard dilutions and IL-23 sample concentration determined.
- >Prepare all reagents and working standards as directed in the previous sections.
- Determine the number of microwell strips required to test the desired number of samples plus appropriate number of wells needed for running blanks and standards. Remove extra microwell strips from holder and store in foil bag with the desiccant provided at 2-8°C sealed tightly.
- Add 100 µl of Standard, control, or sample, per well, then add 50 µl of the working solution of Biotin-Conjugate to each well. Cover with the adhesive strip provided and incubate 2 hours at RT. Adequate mixing is very important for good result. Use a mini-vortexer at the lowest frequency.
- Aspirate each well and wash, repeating the process three times for a total of four washes. Wash by filling each well with Wash Buffer (350µL) using a squirt bottle, manifold dispenser or auto-washer. Complete removal of liquid at each step is essential to good performance. After the last wash, remove any remaining Wash Buffer by aspirating or decanting. Invert the plate and blot it against clean paper towels.
- Add 100 µl of the working solution of Streptavidin-HRP to each well. Cover with a new adhesive strip and incubate for 30 minutes at RT Avoid placing the plate in direct light.
- Repeat the aspiration/wash.
- Add 100 µl of Substrate Solution to each well. Incubate for 10-20 minutes at RT. Avoid placing the plate in direct light.
- Add 100 µl of Stop Solution to each well. Gently tap the plate to ensure thorough mixing.
- Determine the optical density of each well immediately, using a microplate reader set to 450 nm.(optionally 630nm as the reference wave length; 610-650nm is acceptable)
Please note: All documents above are for reference use only and should not be used in place of the documents included with this physical product. If digital copies are needed, please contact us.
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