Swine TNF-Alpha ELISA Assay
The Swine TNF-Alpha ELISA Assay is For Research Use Only
Size: 1×96 wells
Sensitivity: 15 pg/mL
Dynamic Range: 31.25 – 1000 pg/ml
Incubation Time: 3 hours
Sample Type: Serum, Plasma, Cell Culture
Sample Size: 100 µl
Alternative Names: Porcine Tumor Necrosis Factor alpha, Porcine TNF-a, Porcine TNF-alpha, Swine TNF-a, Swine Tumor Necrosis Factor alpha
Tumor necrosis factor alpha (TNF-alpha), also known as cachectin, is a member of the TNF ligand superfamily and has been designated TNFSF1A. It binds to the same cell surface receptors, and shares some biological functions with TNF-b/TNFSF1B. TNF-alpha inhibits the growth of certain tumors. It also plays a critical role in normal host resistance to infection, serving as an immunomodulator and as a mediator of inflammatory responses. Over-production of TNF has been implicated in a number of pathological conditions, including cachexia, septic shock, and autoimmune disorders. TNF-alpha is produced primarily by activated macrophages. Various other porcine cell types, including NK cells, keratinocytes, vascular smooth muscle cells, and granulosa lutein cells are also known to produce TNF-alpha.
The porcine TNF-alpha gene product is a 232 amino acid (aa) residue type II membrane glycoprotein containing a 35 aa cytoplasmic domain, a 21 aa transmembrane domain and a 178 aa extracellular domain. The 156 aa residue soluble TNF-alpha is released from the C-terminus of the membrane protein by TNF-alpha converting enzyme (TACE, ADAM17), a member of the ADAM (a disintegrin and metalloprotease domain) family of metalloproteases. The biologically active TNF-alpha has been shown to exist as a trimer. Porcine TNF-a is active on mouse cells and shares 89% and 79% aa sequence identity with human and mouse TNF-alpha, respectively. Two distinct TNF receptors, referred to as type I (type B, p55, or TNFRSF1A) and type II (type A, p75, or TNFRSF1B), that specifically bind TNF-alpha and TNF-beta with equal affinities are known. The two TNF receptors share aa sequence homology in their extracellular but not their cytoplasmic domains, suggesting that the two receptors employ different signal transduction pathways. Soluble forms of both types of receptors have been found in human and mouse serum.
These soluble receptors are capable of neutralizing the biological activities of the TNFs and may serve to modulate the activities of TNF. Porcine TNF RI shares 79% and 72% aa homology with the human and mouse TNF RI, respectively.
Swine IFN-gamma ELISA Assay
Human TNF-Alpha ELISA Assay
Mouse TNF-Alpha ELISA Assay
The Eagle Biosciences Swine Tumor Necrosis Factor Alpha (TNF-α) ELISA Assay Kit employs the quantitative sandwich enzyme immunoassay technique. A monoclonal antibody specific for TNF-α has been pre-coated onto a microplate. Standard, control, or sample and the working solution of Biotin-Conjugate are pipetted into the wells. Following incubation and wash steps any TNF-α present is bound by the immobilized antibody and the detection antibody specific for TNF-α is binds to the combination of capture antibody- TNF-α in sample. Following a wash to remove any unbound combination, and enzyme conjugate is added to the wells. Following incubation and wash steps a substrate is added. A colored product is formed in proportion to the amount of TNF-α present in the sample. The reaction is terminated by addition of acid and absorbance is measured at 450nm. A standard curve is prepared from seven TNF-α standard dilutions and TNF-α sample concentration determined.
- Prepare all reagents and working standards as directed in the previous sections.
- Determine the number of microwell strips required to test the desired number of samples plus appropriate number of wells needed for running blanks and standards. Remove extra microwell strips from holder and store in foil bag with the desiccant provided at 2-8°C sealed tightly.
- Add 100 µL of Standard, control, or sample, per well, then add 50 µL of the working solution of Biotin-Conjugate to each well. Cover with the adhesive strip provided and incubate 2 hours at RT. Adequate mixing is very important for good result. Use a mini-vortexer at the lowest frequency.
- Aspirate each well and wash, repeating the process three times for a total of four washes. Wash by filling each well with Wash Buffer (350 µL) using a squirt bottle, manifold dispenser or auto-washer. Complete removal of liquid at each step is essential to good performance. After the last wash, remove any remaining Wash Buffer by aspirating or decanting. Invert the plate and blot it against clean paper towels.
- Add 100 µL of the working solution of Streptavidin-HRP to each well. Cover with a new adhesive strip and incubate for 30 minutes at RT Avoid placing the plate in direct light.
- Repeat the aspiration/wash.
- Add 100 µL of Substrate Solution to each well. Incubate for 10-20 minutes at RT. Avoid placing the plate in direct light.
- Add 100 µL of Stop Solution to each well. Gently tap the plate to ensure thorough mixing.
- Determine the optical density of each well immediately, using a microplate reader set to 450 nm.(optionally 630nm as the reference wave length; 610-650nm is acceptable)
Please note: All documents above are for reference use only and should not be used in place of the documents included with this physical product. If digital copies are needed, please contact us.
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