Tumor necrosis factor-alpha (TNF-alpha, or TNF) is secreted by macrophages in response to inflammation, infection and cancer.1 Human Tumor Necrosis Factor (TNF) and Lymphotoxin (TNF-beta) are cytotoxic proteins which have similar biological activities and share 30% amino acid homology.2 TNF-alpha is produced by monocytes, which can stimulate endothelial cells to produce the multilineage growth factor granulocyte-macrophage colony-stimulating factor and extend the role of this immunoregulatory protein to the regulation of hematopoiesis in vitro.3 TNF is a soluble protein that causes damage to tumor cells but has no effect on normal cells. Human TNF has been purified to apparent homogeneity as a 17.3-kilodalton protein from HL-60 leukemia cells and has showed cytotoxic and cytostatic activities against various human tumor cell lines. The human TNF cDNA is 1585 base pairs in length and encodes a protein of 233 amino acids. The mature protein begins at residue 77, leaving a long leader sequence of 76 amino acids.4 TNF-alpha has been mapped to human chromosome 6.2 The standard product used in this kit is recombinant rat TNFα, consisting of 157 amino acids with the molecular mass of 17kD.
Principle of the Assay:The Tumor Necrosis Factor alpha (TNFa), BioAssay™ ELISA Kit (Rat) is based on standard sandwich enzyme-linked immunosorbent assay technology. A mouse monoclonal antibody specific for TNFa has been pre-coated onto 96-well plates. Standards (E. coli-expressed recombinant fragment corresponding to L80 thru L235 of rat TNFa) and test samples are added to the wells, incubated and then biotinylated detection goat polyclonal antibody specific for TNFa is added, followed by an incubation step and a washing step with PBS or TBS buffer. Avidin-Biotin-Peroxidase Complex is added and unbound conjugates are washed away with PBS or TBS buffer. HRP substrate TMB is used to visualize the HRP-mediated reaction. TMB is catalyzed by HRP to produce a blue colored product that changes into yellow upon addition of acidic stop solution. The intensity of the yellow color is proportional to the amount of rat TNFa in the sample.
This kit is intended for the quantitative determination of rat TNFa in cell culture supernatants, cell lysates, tissue homogenates, serum and plasma (heparin, EDTA).
Storage and Stability:Unopened kit is stable at 4°C for 6 months.
Assay Summary:1. Add samples and standards and incubate the plate at 37°C for 90 min. Do not wash. 2. Add biotinylated antibody and incubate the plate at 37°C for 60 min. Wash plate 3 times with 0.01M TBS or PBS
3. Add ABC working solution and incubate the plate at 37°C for 30min. Wash plate 5 times with 0.01M TBS or PBS
4. Add TMB color developing agent and incubate the plate at 37°C in dark for 20-25 min
5. Add Stop solution and read absorbance at 450nm.
