The prototype ligand of the TNF superfamily, TNF-alpha/TNFSF1A, is a pleiotropic cytokine that plays a central role in inflammation and apoptosis. It is synthesized as a 26kD, type II transmembrane protein that is 233aa in length. It contains a 30aa cytoplasmic domain, a 26aa transmembrane segment, and a 177aa extracellular region. TNF-alpha is assembled intracellularly to form a transmembrane, non-covalently-linked homotrimeric protein. Human cells known to express TNF-alpha 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.
The Human TNF-alpha BioAssay™ ELISA Kit is an enzyme-linked immunosorbent assay for the quantitative detection of Human TNF-alpha concentrations in cell culture supernatants, serum, plasma, and tissue.
Sensitivity:4pg/ml
Range:15.625-1000pg/ml
Specificity:Recognizes human IFN-alpha. There was no detectable cross reactivity with any of the tested proteins following IL-1,2,3,4,5,6, 8,10,12,VEGF,TGF,TNF and ICAM.
Sample Volume:100ul/well
Test Principle:This assay employs the Sandwich immunoassay technique. An anti-h TNF-alpha monoclonal coating antibody is adsorbed onto microwells. TNF-alpha present in the sample or standard binds to antibodies adsorbed to the microwells. Following incubation unbound sample or standard are removed during a wash step. a Biotinylated anti-h TNF-alpha antibody is added and binds to TNF-alpha captured by the first antibody. Following incubation unbound Biotinylated anti-h TNF-alpha antibody is removed during a wash step. A Streptavidin-HRP is added and binds to Biotinylated anti-h TNF-alpha antibody. Following incubation unbound Streptavidin-HRP is removed during a wash step. A colored product is formed in proportion to the amount of TNF-alpha 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-alpha standard dilutions and TNF-alpha sample concentration determined.
Storage and Stability:Store powder at 4°C liquid at -20°C. Store other components at 4°C. Stable for at least 6 months For maximum recovery of product, centrifuge the original vial after thawing and prior to removing the cap.
