Tumor necrosis factor-alpha(TNF-alpha, or TNF) is secreted by macrophages in response to inflammation, infection and cancer. Human Tumor Necrosis Factor(TNF) and Lymphotoxin(TNF-beta) are cytotoxic proteins which have similar biological activities and share 30% amino acid homology. 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. 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. TNF-alpha has been mapped to human chromosome 6.

典型操作程序：

1 准备试剂和标准品

2 每孔添加100ul样品和标准品，37℃反应90分钟，不洗。

3 每孔添加100ul生物素标记抗体，37℃反应60分钟。

4 0.01M TBS洗涤3次。

5 每孔添加100ulABC，37℃反应30分钟。

6 0.01M TBS洗涤5次。

7 TMB37℃避光反应30分钟以内。

8 加入TMB终止液，读数。