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. Human cells known to express TNF-α include B cells, colonic columnar epithelial cells, NK and CD3+CD56+ hepatic natural T cells, macrophages, monocytes and monocyte-derived dendritic cells, CD4+ and CD8+ T cells, mast cells, neutrophils, keratinocytes, plasma cells, and adipocytes.
It is synthesized as a 26 kDa, type II transmembrane protein that is 233 amino acids (aa) in length. It contains a 30 aa cytoplasmic domain, a 26 amino acids transmembrane segment, and a 177 amino acids extracellular region. TNF-α is assembled intracellularly to form a transmembrane, non-covalently-linked homotrimeric protein. The 157 aa residue soluble form of TNF-α (sTNF-α is released from the C-terminus of the transmembrane protein through the activity of TNF-α-converting enzyme (TACE), a membrane-bound disintegrin metalloproteinase).
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- 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.
TNF-α has been implicated in a number of pathophysiological processes. It is associated with unregulated pro-inflammatory activity and is thought to be a critical mediator of endotoxin-induced septic shock. Cachexia (or whole body wasting) has also been associated with long-term circulating TNF-α. Other disorders with potential TNF-α involvement include asthma), type 2 diabetes, Crohn’s disease, and rheumatoid arthritis.
|SYNONYMS||DIF-alpha, TNFA, TNFSF2, TNLG1F, TNF|
|Kit Components||96 Wells Quantity/Size|
|Aluminium pouches with a Microwell Plate coated with monoclonal antibody to human TNF-α(8﹡12)||1 plate|
|Human TNF-α Standard lyophilized, 4000 pg/ml upon reconstitution||2 vials|
|Concentrated Biotin-Conjugate anti-human TNF-αmonoclonal 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 7pg/mL.|
|ASSAY RANGE||15.6 - 1000 pg/mL|
|SPECIFICITY||This assay recognizes both natural and recombinant human TNF-α. The factors listed below were prepared at 70ng/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 human||TNF-β, GM-CSF, s TNF RI, s TNF RII/Fc, s TNF RII|
|Recombinant mouse||TNF-α, TNF-α（truncayed）, s TNF RI, s TNF RII IL-4|
Data Analysis Assistance
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