Intended Use:This 2.5 hour ELISpot kit is developed to detect and visualize of single cells secreting human IL-10.
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-10 binds to antibody coated ELISpot plate. ELISpot plate coated with human IL-10 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-10.
Background:The mature human IL-10 is a protein with 160 amino acids and the functional IL-10 is a homodimer. IL-10 is expressed by a variety of cells, including mouse T cells (TH1, TH2, and Tr1 subsets), B cells derived from peripheral blood, tonsils or spleen, Epstein–Barr virus (EBV)-transformed B cell lines, Burkitt’s lymphoma, AIDS lymphomas, monocytes, placental trophoblasts, bronchial epithelial cells, and certain tumor cells including melanomas and carcinomas of various origin.
IL-10 plays an important role in inflammatory and immune responses. The biological activities of IL-10 include both immunosuppressive and immuno-stimulatory effects. For example, IL-10 can suppress the production of pro-inflammatory cytokines by monocytes and neutrophils, and down regulates the expression of activating and co-stimulatory molecules on monocytes and dendritic cells. IL-10 also can improve the growth of B cells and mast cells, and inhibit or enhance the activities of T cells depending on their activation conditions. The different polymorphisms in the IL-10 gene promoter have been demonstrated the association with both SIDS (sudden infant death syndrome) and sudden unexpected death due to infection.
Based on the functions of IL-10, it has been suggested that an IL-10 antagonist can be applied to boost anti-viral immunity against viruses such as EBV and that the IL-10 molecule itself may be used as an anti-inflammatory reagent.
Kit Components:A. Microtiter Plates 1x96wells PVDF-bottom Immunospot plates pre-coated with mouse anti-human IL-8 monoclonal antibody. B. Positive Control 1x1 vial Supplied as a lyophilized recombinant human IL-10. Reconstitute with 250 ul Cell Culture Media before use. Use in 1 hour. C. Wash Buffer (20x)1x 60ml. Add 1 volume of Wash Buffer (20x) to 19 volume of ddH2O. Use in 1 week. Stored at room temperature
D. IL-10 (Biotin) 1x11ml Biotinylated mouse anti-human IL-10 monoclonal antibody. Ready to use. E. Streptavidin (AP)(100x)1x120ul. Add 1 volume of Concentrated Streptavidin 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.
