Intended Use:This 2.5 hour ELISpot kit is developed to detect and visualize of single cells secreting human IL-2.
Principle:Add stimulated cells or cells and stimulant to the wells and incubate at 37°C in CO2 incubator for a specified period. Secreted human IL-2 binds to antibody coated microtiter plate
microtiter plate coated with human IL-2 Capture Antibody. Cells and unbounded proteins are washed away. Streptavidin-AP or Streptavidin-HRP is added and binds to the biotinylated detection antibody. Unbounded proteins are washed away. Biotinylated detection antibody is added and binds to the secreted human IL-2. Substrate Solution is added. A colored precipitate forms and appears as spots at the sites of human IL-2 secreting location. Each individual spot representing an individual human IL-2 secreting cell.
Background:Interleukin 2 (IL-2) is a lymphokine synthesized and secreted primarily by T helper lymphocytes that have been activated by stimulation with certain mitogens or by interaction of the T cell receptor complex with antigen/MHC complexes on the surfaces of antigen-presenting cells. The response of T helper cells to activation is induction of the expression of IL-2 and receptors for IL-2 and, subsequently, clonal expansion of antigen-specific T cells. At this level IL-2 is an autocrine factor, driving the expansion of the antigen-specific cells. IL-2 also acts as a paracrine factor, influencing the activity of other cells, both within the immune system and outside of it. B cells and natural killer (NK) cells respond to IL-2 when properly activated. The so-called lymphocyte activated killer, or LAK cells, appears to be derived from NK cells under the influence of IL-2.
Human IL-2 is a glycoprotein with an apparent molecular weight of 15,000-18,000. Natural IL-2 is glycosylated and varying degrees of glycosylation apparently account for the observed range of molecular weights seen on SDS-PAGE. Human IL-2 is synthesized as a polypeptide of 153 amino acid residues. The first 20 amino acids represent a signal sequence that is cleaved to produce the mature factor. The mature protein contains three cysteine residues, two of which form a disulfide bond that is required for biological activity. Murine IL-2 is approximately 63% identical to human IL-2, but contains a unique stretch of repeated glutamine residues. There is marked species cross-reactivity as human IL-2 has been found to be active on murine cell lines. Cells known to produce IL-2 include thymocytes, gamma delta T cells, B cells, CD4+ and CD8+ T cells, and neurons plus astrocytes.
IL-2 is a factor produced and secreted primarily by activated T helper cells that acts as a autocrine factor driving the expansion of antigen-specific cells and as a paracrine factor influencing the activity of a number of other cells including B cells, NK cells and LAK cells. A simplified but useful view of these activities is of lymphocytes expanding under the influence of IL-2 and becoming the target of other cytokines that cause their functional differentiation
With respect to the specific role of IL-2 on the differentiation of T cells, the separation of CD4+ T helper cells into the categories of TH1 and TH2 according to their function in cell mediated or humoral immunity is a concept that is proving useful. In this system each category of cells secretes a characteristic set of cytokines that functions as a network to push the system either towards cellular immunity (delayed type hyper-sensitivity and cellular cytotoxicity) associated with TH1, or towards humoral immunity (antibody-mediated) associated with TH2. IL-2, along with IFN-gamma and TNF-beta, is a defining product of the TH1 subset. Although the TH1 and TH2 subsets are relatively clearly defined in the murine immune system, these categories are not so clear-cut in the human immune system where the designations TH1-like and TH2-like have been suggested.
Other cells under the possible influence of IL-2 are neutrophils, monocytes, and gamma delta T cells, all of which demonstrate either activation, augmented function, or increased survival when exposed to IL-2. Finally, it should be mentioned that IL-2 is finding its way, along with many other cytokines, into the neurosciences as a possible neuromodulator and growth regulator of glial cells. Because of the central role of the IL-2/IL-2R system in mediation of the immune response, it is obvious that monitoring and manipulation of this system has important diagnostic and therapeutic implications. IL-2 has shown promise as an anti-cancer drug by virtue of its ability to stimulate the proliferation and activities of tumor-attacking LAK and TIL (tumor-infiltrating lymphocytes) cells. However, problems with IL-2 toxicity are still of concern and merit investigation.
Kit Components:A. Microtiter Plates 1x96wells PVDF-bottom Immunospot plates pre-coated with mouse anti-human IL-2 monoclonal antibody. B. Positive Control 1x1 vial. Supplied as a lyophilized recombinant human IL-2. Reconstitute with 250 ul Cell Culture Media before use. Use in 1 hour. C. Wash Buffer (20x) 1x60ml. Add 1 volume of Wash Buffer (20x) to 19 volume of ddH2O. Use in 1 week. Stored at room temperature. D. IL-2 (Biotin)1x11ml Biotinylated mouse anti-human IL-2 monoclonal antibody. Ready to use. E. Streptavidin (AP)(100x) 1x120ul. Add 1 volume of Streptavidin (AP)(100x) to 100 volumes of F. (Streptavidin (AP) Diluent) before use. Use in 1 month. Stored at 4°C. F. Streptavidin (AP) Diluent 1x11ml. Ready to use
G. BCIP/NBT Substrate Solution 1x11ml. Ready to use.
Storage and Stability:Store other components at 4ºC. Stable for at least 6 months. For maximum recovery of product, centrifuge the original vial after thawing and prior to removing the cap.
