Tumor Necrosis Factor alpha (TNF-a), also known as cachectin was initially named for its remarkable ability to cause hemorrhagic necrosis of tumors in mice. The TNF-a gene located on chromosome 6 encodes a 233aa prohormone bound to the plasma membrane, with the mature form (157aa, 17kD) exposed on the extracellular surface. Soluable, mature TNF-a is released upon cleavage of the C-terminal. The primary source in vivo of TNF-a is thought to be the monocyte/macrophage but various cell types are known to express this cytokine such as lymphocytes, basophils, eosinophils, mast cells, NK cells, T cells, B cells, kerantinocytes, Kupffer cells, astrocytes, and some types of tumors. TNF-a is produced upon stimulation with cytokines such as IL-1, 1L-2, GM-CSF, TNF-a itself and with bacterial lipopolysaccharide (LPS) which is a potent inducer. TNF-a once produced and secreted will bind to TNF-a pleiotropic cytokine that can induce disease through TNF-a toxicity (tissue injury, catabolic illness, and mediating shock) and improve host defence mechanisms (stimulating inflammation and increasing immune cell function). In the future, therapies may be developed by blocking TNF-a harmful effects and enhancing TNF-a beneficial effects.
This TNF-a enzyme linked immunosorbent assay (ELISA) applies a technique called a quantitative sandwich immunoassay. The microtiter plate provided in this kit has been pre-coated with a monoclonal antibody specific to TNF-a. Standards or samples are then added to the appropriate microtiter plate wells with a biotin-conjugated polyclonal antibody preparation specific for TNF-a and incubated. TNF-a if present, will bind and become immobilized by the antibody pre-coated on the wells and then be "sandwiched" by biotin conjugate. In order to measure the concentration of TNF-a in the samples, this kit includes two calibration diluents (Calibrator Diluent I for serum/plasma testing and Calibrator Diluent II for cell culture supernatant testing.) According to the testing system, the provided standard is diluted (2-fold) with the appropriate Calibrator Diluent and assayed at the same time as the samples. This allows the operator to produce a standard curve of Optical Density (O.D) versus TNF-a concentration (pg/ml). The concentration of TNF-a in the samples is then determined by comparing the O.D. of the samples to the standard curve.
Storage and Stability:Store kit components at 4°C except *T9160-01D. Stable for 6 months. Store T9160-01D lyophilized powder at 4°C. Store reconstituted stock at -20°C for <30 days. For maximum recovery of product, centrifuge the original vial prior to removing the cap. Further dilutions can be made in assay buffer.
