IL-12/IL-23 p40, Human, BioAssay(TM) ELISpot Kit

Interleukin 12 (IL-12), also known as natural killer cell stimulatory factor (NKSF) or cytotoxic lymphocyte maturation factor (CLMF), is a heterodimeric pleiotropic cytokine made up of a 40kD (p40) subunit and a 35kD (p35) subunit. The IL-12 p40 subunit is shared by IL-23, another heterodimeric cytokine that has biological activities similar to, as well as distinct from, IL-12. IL-12 is produced by macrophages and B cells and has been shown to have multiple effects on T cells and natural killer (NK) cells. While mouse IL-12 is active on both human and mouse cells, human IL-12 is not active on mouse cells. Intended Use: For the quantitative determination of the frequency of cells releasing human IL-12/IL-23 p40. Test Principle: The enzyme-linked immunospot (ELISpot) assay was originally developed for the detection of individual B cells secreting antigen-specific antibodies (31, 32). This method has since been adapted for the detection of individual cells secreting specific cytokines or other antigens (33, 34). ELISpot assays employ the quantitative sandwich enzyme-linked immunosorbent assay (ELISA) technique. A monoclonal antibody specific for human IL-12/IL-23 p40 has been pre-coated onto a PVDF (polyvinylidene difluoride)-backed microplate. Appropriately stimulated cells are pipetted into the wells and the microplate is placed into a humidified 37 degrees C CO2 incubator for a specified period of time. During this incubation period, the immobilized antibody in the immediate vicinity of the secreting cells bind secreted IL-12/IL-23 p40. After washing away any cells and unbound substances, a biotinylated polyclonal antibody specific for human IL-12/IL-23 p40 is added to the wells. Following a wash to remove any unbound biotinylated antibody, alkaline-phosphatase conjugated to streptavidin is added. Unbound enzyme is subsequently removed by washing and a substrate solution (BCIP/NBT) is added. A blue-black colored precipitate forms and appears as spots at the sites of cytokine localization, with each individual spot representing an individual IL-12/IL-23 p40 secreting cell. The spots can be counted with an automated ELISpot reader system or manually using a stereomicroscope. Kit Components: 1. Human IL-12/IL-23 p40 Microplate, 1x96wells 2. Detection Antibody Concentrate, 1x150ul 3. Streptavidin-AP Concentrate A, 1x150ul 4. Dilution Buffer 1, 1x15ml 5. Dilution Buffer 2, 1x12ml 6. Wash Buffer Concentrate, 1x50ml 7. BCIP/NBT Chromogen, 1x12ml 8. Human IL-12/IL-23 p40 Positive Control, 1x1vial Storage and Stability: Store other components at 4 degrees C. Stable for at least 6 months For maximum recovery of product, centrifuge the original vial after thawing and prior to removing the cap.