Intended Use:This 2.5 hour ELISpot kit is developed to detect and visualize of single cells secreting human IL-6.
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-6 binds to antibody coated microtiter plate
microtiter plate coated with human IL-6 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-6. Substrate Solution is added. A colored precipitate forms and appears as spots at the sites of human IL-6 secreting location. Each individual spot representing an individual human IL-6 secreting cell.
Background:Interleukin 6 (IL-6) is a multifunctional protein that plays important roles in host defense, acute phase reactions, immune responses, and hematopoiesis. (1) Synonyms for IL-6 include: B cell stimulatory factor-2 (BSF-2), (2) hybridoma/plasmacytoma growth factor, (3) hepatocyte stimulating factor, cytotoxic T cell differentiation factor and macrophage-granulocyte inducing factor 2A (MGI-2A).(1) IL-6 is expressed by a variety of normal and transformed cells including T cells, B cells, monocytes/macrophages, fibroblasts, hepatocytes, keratinocytes ,astrocytes, vascular endothelial cells, mesangial cells, osteoblasts, carcinomas, sarcomas, myelomas, glioblastomas, and melanomas. The production of IL-6 is up-regulated by numerous signals including mitogenic or antigenic stimulation, lipopolysaccharide, calcium ionophore, IL-1, IL-2, IFN, TNF, PDGF, and viruses. IL-4 and IL-13 inhibit IL-6 expression in monocytes. (4-6)
Natural human and murine IL-6 are glycoproteins containing N-and/or O-linked carbohydrates (human IL-6 contains two potential N-glycosylation sites, while mouse IL-6 has none). In comparison with mouse IL-6, human IL-6 exhibits approximately 65% sequence homology at the nucleotide level, and 42% homology at the amino acid level. Although human and mouse IL-6 are equally active on mouse cells, mouse IL-6 is not active on human cells
IL-6 is predicted to have a four helix-bundle type tertiary structure found in a number of other cytokines including growth hormone, EPO, G-CSF, OSM, IL-11, CNTF, LIF, MGF, Prolactin, etc. (7-9) The gene structures of these cytokines also show varying degrees of relatedness. Based on these criteria, it has been suggested that these cytokines evolved from a common ancestral gene. Results of structure-function studies of IL-6 and other four α-helix bundle cytokines indicated that the c-terminal (helix D) regions of these cytokines are primarily responsible for binding to the receptors. (1. 4)
Interleukin 6 exerts multiple functions on numerous target cells. IL-6 plays an important role in immune functions. It has effects on B cell differentiation and antibody production, on cytotoxic T cell differentiation, on T cell activation, growth and differentiation, and on the induction of IL-2Rα chain expression and IL-2 production in T cells. (11-13) In hemopoiesis, IL-6 has blast cell growth factor activity and can synergize with IL-3 to shorten the G0 period of early hemopoietic progenitors. 14) In addition, IL-6 has been found to synergize with IL-3 in megakaryocyte development, increasing platelet numbers in vivo and the number, size and average ploidy value of megakaryocyte colonies formed from mouse or human bone marrow cells in vitro. (15-18) Similarly to IL-11 and LIF, IL-6 can induce the synthesis of hepatic acute phase proteins both in vivo and in vitro. (19) IL-6 has growth factor activities and will stimulate the growth of hybridomas, plasmacytomas, myelomas, sarcomas, (20, 21) carcinomas, (22) EBV-transformed B cells, (23) keratinocytes, and mesangial cells. In contrast to its growth stimulatory activities, IL-6 is also a growth inhibitor for a number of leukemia and carcinoma cell lines. Additional bioactivities attributed to IL-6 include: induction of terminal differentiation of M1 myeloid leukemic cells; (24) the differentiation and survival of neuronal cells; (25, 26) and the activation of osteoclast development. (10) Although IL-6 was also discovered as an antiviral factor produced by human diploid fibroblasts, the question of whether or not IL-6 has antiviral activity is controversial. Many groups have been consistently unable to find any antiviral activity for recombinant human IL-6. (1)
The various activities of IL-6 described above suggest that this factor will have a major role in the mediation of the inflammatory and immune responses initiated by infection or injury. Although the exact functions of IL-6 in vivo are not known, elevated IL-6 levels have been reported to be associated with a variety of diseases, including auto-immune diseases, mesangial proliferative glomerulonephritis, psoriasis, and malignancies such as plasmacytoma and myeloma. A great deal of work is currently in progress in order to provide a better understanding of the role of IL-6 in the modulation of normal and pathological processes.
B. Positive Control 1x1 vial Supplied as a lyophilized recombinant human IL-6. 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-6 (Biotin) 1x11ml. Biotinylated mouse anti-human IL-6 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.
