Interleukin 2, Primate, BioAssay(TM) ELISpot Kit (IL-2)

Interleukin 2 (IL-2) was initially identified as a T cell growth factor that is produced by T cells following activation by mitogens or antigens. Since then, it has also been shown to stimulate the growth and differentiation of B cells, natural killer (NK) cells, lymphocyte activated killer (LAK) cells, monocytes/macrophages and oligodendrocytes. At the amino acid sequence level, there is approximately 60% - 90% similarity between species. Mature human IL-2 shows 65%, 67%, 72%, 78%, and 64% aa identity to mouse, rat, pig, cat, and cow IL-2, respectively. Intended Use: For the quantitative determination of the frequency of cells releasing primate IL-2. Test Principle: The enzyme-linked immunospot (ELISpot) assay was originally developed for the detection of individual B cells secreting antigen-specific antibodies (21, 22). This method has since been adapted for the detection of individual cells secreting specific cytokines or other antigens (23, 24). ELISpot assays employ the quantitative sandwich enzyme-linked immunosorbent assay (ELISA) technique. A monoclonal antibody specific for primate IL-2 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-2. After washing away any cells and unbound substances, a biotinylated polyclonal antibody specific for primate IL-2 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-2 secreting cell. The spots can be counted with an automated ELISpot reader system or manually using a stereomicroscope. Kit Components: 1. Precoated Microplate, 1x96 wells 2. Detection Antibody Concentrate, 1x150ul 3. Streptavidin-AP Concentrate A, 1x150ul 4. Dilution Buffer 1, 1x12ml 5. DIlution Buffer 2, 1x12ml 6. Wash Buffer Concentrate, 1x50ml 7. BCIP/NBT Chromogen, 1x12ml 8. Primate IL-2 Positive Control, 1x1vial Storage and Stability: Store 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.