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 17.3kD 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).
Intended Use:TNF alpha BioAssay™ ELISA Kit, Human is a sandwich ELISA for quantitative detection of human TNF alpha in cell culture supernates, serum and plasma (heparin, EDTA, citrate).
Assay Summary:1. Add samples and standards and incubate the plate at 37°C for 90 minutes. Do not wash. 2. Add biotinylated antibodies and incubate the plate at 37°C for 60 minutes. Wash plate 3 times with 0.01M TBS
3. Add ABC working solution and incubate the plate at 37°C for 30 minutes. Wash plate 5times with 0.01M TBS
4. Add TMB color developing agent and incubate the plate at 37°C in dark for 25-30 minutes
5. Add TMB stop solution and read..
Storage and Stability:Store *144065B powder at 4°C. Once reconstituted store at 4°C for up to 12 hours or at -20°C for up to 48 hours. Store other components at 4°C. Stable for 6 months after receipt. For maximum recovery of product, centrifuge the original vial after thawing and prior to removing the cap.
