Tumor Necrosis Factor alpha (TNF-a) is a multifunctional proinflammatory cytokine, mainly secreted by activated macrophages. The cytokine was named for its remarkable ability to cause hemorrhagic necrosis of tumors in mice. It is implicated with a variety of biological procedures including systemic inflammation, cell proliferation, apoptosis, lipid metabolism, and coagulation. It is well documented that TNF-a functions through its receptors, TNFR1 (p55) and TNFR2 (p75). TNF-a plays an important role in the immune response to bacterial, and certain fungal, viral, parasitic invasions, in tissue remodeling, autoimmune-diseases and the necrosis of specific tumors. The pleiotropic effect of TNF-a regulation is attributed to its ability to trigger multiple signaling pathways simultaneously. TNF-a hyper-expression in response to the components of some bacteria such as LPS can cause life threatening septic shock. Recombinant TNF-a, in combination with chemotherapy, has been applied for synergistic treatment of soft sarcomas, melanomas and other irresectable tumors. Anti-TNF-a therapy has been used for treatment of rheumatoid arthritis.
Intended Use:This Mouse TNF-alpha (a) ELISA Kit is to be used for the in vitro quantitative determination of mouse tumor necrosis factor alpha (mouse TNF-a) concentrations in serum and cell culture supernatant.
Sensitivity:<4.5pg/ml
Range:15.6-1000pg/ml
Test Principle:This mouse TNF-a 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 antibody specific to mouse TNF-a. Standards or samples are then added to the appropriate microtiter plate wells with a biotin-conjugated monoclonal antibody preparation specific for mouse TNF-a and incubated. Mouse TNF-a if present, will bind and become immobilized by the antibody pre-coated on the wells and then be “sandwiched” by biotin conjugate. The microtiter plate wells are thoroughly washed to remove unbound mouse TNF-a and other components of the sample. In order to quantify the amount of mouse TNF-a present in the sample, avidin conjugated to Horseradish Peroxidase (HRP) is added to each microplate well and incubated. Avidin is a tetramer containing four identical subunits that each has a high affinity-binding site for biotin. The wells are thoroughly washed to remove all unbound HRP-conjugated Avidin and 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 mouse TNF-a, biotin-conjugated antibody, and enzyme-conjugated Avidin 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 TNF-a in the samples, this kit includes two calibration diluents (Calibrator Diluent I for serum/plasma testing and Calibrator Diluent II for cell culture supernatant testing.) According to the testing system, the provided standard is diluted (2-fold) 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 TNF-a concentration (pg/ml). The concentration of mouse TNF-a in the samples is then determined by comparing the O.D. of the samples to the standard curve.
Kit Components:MOUSE TNf-a MICROTITER PLATE BIOTIN CONJUGATE AVIDIN CONJUGATE MOUSE TNF-a 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.
