Principle of the Assay
This assay employs the quantitative sandwich enzyme immunoassay technique. A monoclonal antibody specific for TNF-α has been pre-coated onto a microplate. Standards and samples are pipetted into the wells and any TNF-α present is bound by the immobilized antibody. Following incubation unbound samples are removed during a wash step, and then a detection antibody specific for TNF-α is added to the wells and binds to the combination of capture antibody-TNF-α in sample. Following a wash to remove any unbound combination, and enzyme conjugate is added to the wells. Following incubation and wash steps a substrate is added. A coloured product is formed in proportion to the amount of TNF-α present in the sample. The reaction is terminated by addition of acid and absorbance is measured at 450nm. A standard curve is prepared from seven TNF-α standard dilutions and TNF-α sample concentration determined.
For Use with serum, plasma and cell culture supernatants. For Research Use Only. Not for use in diagnostic procedures.
The prototype ligand of the TNF superfamily, TNF-α/TNFSF1A, is a pleiotropic cytokine that plays a central role in inflammation and apoptosis. TNF-α is produced by activated macrophages and other cell types including T and B cells, NK cells, LAK cells, astrocytes, endothelial cells, smooth muscle cells and some tumor cells.
Mouse TNF-α cDNA encodes a 235 amino acid (aa) residue type II membrane protein. The 156 aa residue soluble TNF-α is released from the C-terminus of the membrane-anchored TNF-α by proteolytic cleavage by a matrix metalloprotease.
The membrane-anchored form of TNF-α has been shown to have lytic activity and may also have an important role in intercellular communication. The biologically active TNF-α has been shown to exist as a trimer. TNF-α is reported to promote inflammatory cell infiltration by upregulating leukocyte adhesion molecules on endothelial cells, serve as a chemotactic agent for monocytes, and activate phagocyte killing mechanisms. Deficiencies in either TNF-α or its receptors can increase susceptibility to infection by intracellular pathogens. TNF-α may also play a role in lymphoid tissue development. Knockout mice lack splenic B cell follicles and the ability to form germinal centers. Other potential physiological roles for TNF-α and its receptors include regulating the differentiation of hematopoietic stem and progenitor cells.
|SYNONYMS||DIF, TNF-a, TNF-alpha, TNFSF2, TNFalphaa, Tnfsf1a, Tnlg1f, Tnf|
|Kit Components||96 Wells Quantity/Size|
|Aluminium pouches with a Microwell Plate coated with monoclonal antibody to mouse TNF-α(8﹡12)||1 plate|
|Mouse TNF-α Standard lyophilized, 1000 pg/ml upon reconstitution||2 vials|
|Concentrated Biotin-Conjugate anti-mouse TNF-α monoclonal antibody||2 vials|
|Streptavidin-HRP solution||2 vials|
|Standard /sample Diluent||1 bottle|
|Biotin-Conjugate antibody Diluent||1 bottle|
|Streptavidin-HRP Diluent||1 bottle|
|Wash Buffer Concentrate 20x (PBS with 1% Tween-20)||1 bottle|
|Substrate Solution||1 vial|
|Stop Solution||1 vial|
|Adhesive Films||4 pieces|
|Product data sheet||1 copy|
|Storage||Store at 2 - 8°C|
|REPEATABILITY||The coefficient of variation of both intra-assay and inter-assay were less than 10%.|
|SENSITIVITY||The minimum detectable dose was 3.9pg/mL.|
|ASSAY RANGE||7.8 - 500 pg/mL|
|SPECIFICITY||This assay recognizes both natural and recombinant mouse TNF-α. The factors listed below were prepared at 50ng/ml in Standard /sample Diluent and assayed for cross-reactivity and no significant cross-reactivity or interference was observed.|
Factors assayed for cross-reactivity
|Recombinant mouse||G-CSF, GM-CSF, C10, IFN-γ, IL-1α, IL-1β, IL-2 , IL-3, IL-5, IL-6, IL-7, IL-9, LIF, M-CSF, SCF, VEGF, TNF-β|
|Recombinant human||TNF-α, TNF sRI, TNF sRII|
Data Analysis Assistance
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