Intended Use:This 2.5 hour ELISpot kit is developed to detect and visualize of single cells secreting human IL-2sRa.
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-2sRa binds to antibody coated microtiter plate
microtiteer plate coated with human IL-2sRa 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-2sRa. Substrate Solution is added. A colored precipitate forms and appears as spots at the sites of human IL-2sRa secreting location. Each individual spot representing an individual human IL-2sRa secreting cell.
Background:The biological function of IL-2 is obtained by binding to the specific interleukin-2 receptor (IL-2R). The IL-2R consists of three non-covalently linked chains, all of which are type I transmembrane proteins and include the α chain (IL-2Rα, p55), β chain (IL-2Rβ, p75), and γ chain (IL-2Rγ, p65). The α chain is cleaved from the cell surface via nonspecific proteolysis.
IL-2Rα and IL-2Rβγ dimers bind to different residues on the IL-2 protein. The IL-2Rα complex displays low affinity and the IL-2Rαβ complex displays intermediate affinity for IL-2 binding. Both IL-2Rα and IL-2Rαβ complexes are unable to transduce a signal. The IL-2Rβγ complex has intermediate affinity for IL-2 binding and can transduce a signal with a relatively high concentration of IL-2. The IL-2Rαβγ trimer is the high-affinity receptor for IL-2 and can transduce a signal successfully.
Many cells are capable of expressing IL-2Rα including the antigen-activated T cells and B cells, and approximately 10% of natural killer (NK) cells, leukemia and lymphoma cells. When produced by activated T cells, the α chain is 10-20 folds in excess of the β and γ chain. A soluble IL-2Rα can be detected in tissue culture media of IL-2R+ cells and in the serum of experimental animals and humans undergoing an immune response.
The major biological activities of IL-2R include promoting the proliferative expansion of T cells and NK cells upon activation, promoting the persistence of antigen-selected memory T cells, and promoting homeostasis of the immune system after it has successfully responded to an antigen. However, the biological activity of soluble IL-2Rα is unclear. It has been reported that elevated IL-2 sRα level is accompanied by increased T and B cell activation and immune system activation as observed in rheumatoid arthritis, systemic lupus erythematosis (SLE), some leukemias and lymphomas. Because of its low affinity, IL-2 sRα would be expected to be an inhibitor of IL-2.
Kit Components:A. Microtiter Plates 1x96wells PVDF-bottom Immunospot plates pre-coated with mouse anti-human IL-2sRa monoclonal antibody. B. Positive Control 1x1 vial. Supplied as a lyophilized recombinant human IL-2sRa. 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. Human IL-2sRa (Biotin) 1x11ml Biotinylated mouse anti-human IL-2sRa 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.
