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EK000009-EK0526: Rat TNFα ELISA Kit
Range: 15.6pg/ml-1000pg/ml (body fluids, tissue lysates or cell culture supernates); 7.8pg/ml-500pg/ml (rat sera).
Sensitivity < 1 pg/ml
Specificity: no detectable cross-reactivity with any other cytokine.
Application: for quantitative detection of rat TNFα in sera, plasma, body fluids, tissue lysates or cell culture supernates.
Expiration: four months at 4°C and eight months at -20°C.
Background
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
Principle
The ELISA Kit is based on standard sandwich enzyme-linked immune-sorbent assay technology. The target specific antibodies are precoated onto 96-well plates. The target from the sample is bound to the microwell. The biotinylated target specific detection antibodies are added to the microwells and followed by washing with the PBS or TBS buffer. Avidin-Biotin-Peroxidase Complex is added and unbound conjugates are washed away with the PBS or TBS buffer. TMB, the HRP (horseradish peroxidase) substrate, is used to visualize color change resulting from the HRP enzymatic reaction. TMB is catalyzed by HRP to produce the blue color. The color changes into yellow after the acidic stop solution is added. The density of the yellow color is proportional to the target amount from the sample captured in the microwells.
Reference
1. Brenner, D. A.; O'Hara, M.; Angel, P.; Chojkier, M.; Karin, M. Prolonged activation of JUN and collagenase genes by tumour necrosis factor-alpha. Nature 337: 661-663, 1989.
2. Nedwin, G. E.; Naylor, S. L.; Sakaguchi, A. Y.; Smith, D.; Jarrett-Nedwin, J.; Pennica, D.; Goeddel, D. V.; Gray, P. W. Human lymphotoxin and tumor necrosis factor genes: structure, homology and chromosomal localization. Nucleic Acids Res. 13: 6361-6373, 1985.
3. Broudy, V. C.; Kaushansky, K.; Segal, G. M.; Harlan, J. M.; Adamson, J. W. Tumor necrosis factor type alpha stimulates human endothelial cells to produce granulocyte/macrophage colony-stimulating factor. Proc. Nat. Acad. Sci. 83: 7467-7471, 1986.
4. Wang, A. M.; Creasey, A. A.; Ladner, M. B.; Lin, L. S.; Strickler, J.; Van Arsdell, J. N.; Yamamoto, R.; Mark, D. F. Molecular cloning of the complementary DNA for human tumor necrosis factor. Science 228: 149-154, 1985.
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