IFN-ã is a multifunctional cytokine, possessing important immune-modulation, anti-viral and anti-tumor properties. It is produced by natural killer cells and natural killer T cells, plays an important part in innate immunity. CD4+ T helper 1 cells, CD8 cytotoxic T lymphocyte (CTL) and effecter T cells also produce IFN-y when acquired immunity develops. IFN-ã stimulates its own expression and up-regulates other genes through the Jak-Stat signaling pathway. IFN-y released after viral infection stimulates the production of large amount of protein kinase R, which phosphorylates transcription initiation factor elF in response to viral infection. As a results, enzymes critical to mRNA replication is reduced and mRNA replication inhibited. Experiment with murine cytomegalovirus infected mice (1) showed that natural killer cells are the main source of IFN-y. In natural killer cell depleted mice, viral hepatitis and viral replication levels were higher than control group. The experiment also demonstrated that inhibition of viral replication is mediated by IFN-a, since inhibition is independent of cytolytic activity. In the same study, administration of IL-12 to the MCMV infected mice was found to induce the production of IFN-ã by natural killer cells. Previously (3), interleukin IL-12 has been identified, by Kobayashi et al, as natural killer cell stimulatory factor released from Epstein-Barr-virus infected B cells
IFN-y influences the emergence of cellular immunity through stimulating the maturation of CD4+ T helper 1 cells (Th1) and cytotoxic CD8+ cells (2). IFN-y suppresses T helper 2 differentiation and humoral immunity, and was also found to regulate mouse subclass switching (5). IFN-y is a potent macrophage activator. It binds to macrophage in conjugation with CD40, causing the macrophage to produce elevated amounts of MHCmolecules. It also stimulates the production of antigen-processing associated transporters and enzymes. The accumulation of macrophages and T helper 1 cells around M. tuberculosis infected cells is believed to the mechanism for granuloma formation in TB patients. IFN-y was found to limit infection (4) in chronic granulomatous diseases (CGD). A form of this cytokine has been approved by FDA for treatment of CGD
Inhibition to cancer growth by IFN-y is well documented. Recombinant IFN-y has been used for treatment of various cancer types in combination with chemotherapy. Recent experiment with mouse skin cancer model showed that IFN-y plasmidDNAhad inhibitory effects in skin carcinogenesis (6), indicating IFN-y DNAcould be useful for cancer gene therapy
This mouse IFN-y ELISA is a 4.5 hour solid phase immunoassay readily applicable to measure IFN-y levels in mouse serum, cell culture supernatant, and other biological fluids in the range of 0 to 3200pg/ml. It is expected to be useful for investigations into the relationship between IFN-y and various diseases.
Intended Use:This Mouse IFN-y ELISA kit is to be used for the in vitro quantitative determination of mouse interferon-y concentrations in mouse serum and cell culture supernatant.
Sensitivity:<7.5pg/ml
Range:50-3200pg/ml
Test Principle:This mouse IFN-y enzyme-linked immunosorbent assay (ELISA) applies a technique called a quantitative sandwich immunoassay. The microtiter plate provided in this kit has been pre- coated with a monoclonal specific for mouse IFN-u. Standards or samples are then added to the appropriate microtiter plate wells and incubated. In order to quantitatively determine the amount of mouse IFN-u present in the sample, a biotin-conjugated antibody specific for mouse IFN-y is added to each well to "sandwich" the IFN-u immobilized during the first incubation. The microtiter plate then undergoes a second incubation and the wells are thoroughly washed to remove all unbound Biotin-conjugated antibodies. Avidin can bind to biotin with high affinity and amplify the biotin signal. A preparation of avidin conjugated HRP is added to each well and incubated. The avidin HRP conjugate will bind to wells containing biotin. After washing the wells, the unbounded avidin-HRP conjugate is removed. A TMB (3,3'5, 5' tetramethyl-benzidine) substrate solution is added to each well. The enzyme (HRP) and substrate are allowed to react over a short incubation period. Only those wells that contain IFN-y and biotin-conjugated antibody, avidin conjugated HRP will exhibit a change in color. The enzyme-substrate reaction is terminated by the addition of a sulphuric acid solution and the color change is measured spectrophotometrically at a wavelength of 450nm ± 2nm
In order to measure the concentration of mouse IFN-y in the samples this kit contains two calibration diluents. (Calibrator Diluent I for serum/plasma testing and Calibrator Diluent II for cell culture supernatant). According to the testing system, the provided standard is 2 x fold serial diluted with the appropriate Calibrator Diluent and assayed at the same time as the samples. This allows the operator to produce a standard curve of Optical Density (O.D.) versus mouse IFN-y concentration (pg/ml). The concentration of mouse IFN-y in the samples is then determined by comparing the O.D. of the samples to the standard curve.
Kit Components:MOUSE IFn-y MICROTITER PLATE MOUSE IFn-y BIOTIN CONJUGATE AVIDIN HRP CONJUGATE MOUSE IFn-y STANDARD CALIBRATOR DILUENT I CALIBRATOR DILUENT II WASH BUFFER (20X/96 wells, 30X/192 wells) SUBSTRATE A SUBSTRATE B STOP SOLUTION
Storage and Stability:Store powder at 4°C liquid at -20°C. Store other components at 4°C. Stable for 6 months For maximum recovery of product, centrifuge the original vial after thawing and prior to removing the cap.
