| Interferon gamma (IFN-g) is a multifunctional protein first observed to have antiviral activity in cultures of Sindbis virus-infected human leukocytes stimulated by PHA. (1)) The biochemistry and biological activities of the interferons have been extensively reviewed. Produced by both CD4+ and CD8+ T lymphocytes and natural killer (NK) cells, INF-g is now known to be both an inhibitor of viral replication and a regulator of numerous immunological functions. IFN-g influences the class of antibody produced by B cells up-regulates classes I and II MHC complex antigens and increases the efficiency of macrophage-mediated killing of intracellular parasites. (2, 3) Most of the activities attributed to IFN-g are believed to be mediated by IFN-g-induced proteins. The appearance of such proteins is a consequence of IFN-g binding to a specific receptor that is distinct from the receptor for IFN-a and b. (4) Human IFN-g is reported to be active only on human and non-human primate cells. (5) The biochemistry and biological activities of the interferons have been extensively reviewed. (2-9) |
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| Catalog # | I8449-08Q |
| Human IFN-g is a 143 amino acid residue glycoprotein with MW of 20 or 25kD that demonstrates little sequence homology to IFN-a and b. (10-13) Naturally occurring IFN-g is found as either of two molecular-weight-species, differing in degree of glycosylation. The 25kD species is glycosylated at both potential N-linked glycosylation sites on the molecule (Asn 25 and 97), while the 20kD species is glycosylated only at Asn97. (17, 18) In neither case glycosylation is required for biological activity. (13, 16) Two allelic variants of IFN-g have been described differing by the presence of an Arg or Gln at position 137. (10, 16) |
| Although the cDNA encoding for IFN-g predicts a protein of 146 amino acid residues, the form secreted by mammalian cells shows a truncation of three amino acid residues from the N-terminus and the conversion of the fourth residue from glutamic acid to pyroglutamate. (11) The secreted form of IFN-g has no potential for the formation of disulfide bonds. (13) Human IFN-g apparently exists as a head-to-tail dimer in solution with the C-terminus of one monomer aligned with the N-terminus of the other monomer. (18, 19) |
| IFN-g possesses a variety of functions. Produced by CD8+, NK and TH2 T helper cells, IFN-g has documented antiviral, antiprotozoal and immunomodulatory activities, (20-24) although IFN-a and IFN-b seem to have more potential antiviral activities than IFN-g. (24) The antiprotozoal activity of IFN-g against Toxoplasma and Chlamydia is believed to result from indoleamine 2, 3-dioxygenase activity, an enzyme induced by IFN-g. (25) The immunomodulatory effects of IFN-g are extensive and diverse. |
| In monocyte/ macrophages, IFN-g increases expression of class 1 MHC antigens; increases the production of IL-1, platelet-activating factor, H2O2, and pterin; protects monocytes against LAK cell-mediated lysis; down-regulates IL-8 mRNA expression that is up-regulating TGF-b receptor expression and up-regulating expression of the IL-2Rg subunit. (23, 25, 26-29) It has also been demonstrated to be chemotactic for monocytes but not neutrophils. (30) IFN-g selectively enhances both Ig G2a secretion by LPS-stimulated B cell activation. (31, 32) IFN-g has also been reported to induce its own expression. IFN-g production accompanying local inflammation results in the induction of IFN-g mRNA synthesis at distant sites. This effect could be due to circulating IFN-g or the production of IFN-g by migrating cells (33). IFN-g has also been shown to up-regulate ICAM-1 but not E-selectin or VCAM-1 expression on endothelial cells (34). Finally, IFN-g has recently been implicated in the development of a cholinergic phenotype in embryonic septal neurons. In cultures of rat septal nuclei, IFN-g induced the development of cholinergic neurons. |
| This 2.5 hour ELISpot kit is developed to detect and visualize of single cells secreting human IFN-g. |
| Principle | 1. Add stimulated cells or cells and stimulant to the wells pre-coated with human IFN-g antibody and incubate at 37°C in CO2 incubator for a specified period. Secreted human IFN-g binds to antibody coated ELISpot plate. |
| 2. Cells and unbound proteins are washed away. |
| 3. Biotinylated detection antibody is added and binds to the secreted IFN-g. |
| 4. Unbound proteins are washed away. |
| 5. Streptavidin-AP or Streptavidin-HRP is added and binds to the biotinylated detection antibody. |
| 6. Unbound proteins are washed away. |
| 7. Substrate Solution is added. A colored precipitate forms and appears as spots at the sites of human IFN-g secreting location. Each individual spot representing an individual human IFN-g secreting cell. |
| Kit Components | I8449-08Q1: Microtiter Plate 1x96 wells PVDF-bottom Immunospot plates pre-coated with mouse anti-human IFN-gamma monoclonal antibody. |
| I8449-08Q2: Positive Control 1x1 vial. Supplied as a lyophilized recombinant human IFN-gamma. Reconstitute with 250ul Cell Culture Media before use. Use in 1 hour. The final concentration is 8ng/ml. |
| I8449-08Q3: Wash Buffer (20X) 1x60ml. Add 1 volume of Wash Buffer (20X) to 19 volume of ddH2O. Use in 1 week. If the concentrate is stored ar 4ºC, crystals may form. Allow to reach RT before reconstitution. |
| I8449-08Q4: Mab Detection Antibody (Biotin). 1x11ml. Biotinylated mouse anti-human IFN-gamma monoclonal antibody. Ready to use. |
| I8449-08Q5: Streptavidin (AP) (100X) 1x120ul. Add 1 volume of Streptavidin (AP)(100X) to 100 volumes of Streptavidin (AP) Diluent before use. Use in 1 month. |
| I8449-08Q6: Streptavidin (AP) Diluent 1x11ml. Ready to use. |
| I8449-08Q7: BCIP/NBT Substrate Solution 1x11ml. Ready to use. |
| Storage and Stability | Store all 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. |
| Important Note | This product as supplied is intended for research use only, not for use in human, therapeutic or diagnostic applications without the expressed written authorization of United States Biological. |
