Tumor necrosis factor alpha (TNF-), 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-/TNFSF1B. TNF-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-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-.

The porcine TNF- 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-is released from the C-terminus of the membrane protein by TNF-converting enzyme (TACE, ADAM17), a member of the ADAM (a disintegrin and metalloprotease domain) family of metalloproteases. The biologically active TNF-has been shown to exist as a trimer. Porcine TNF-is active on mouse cells and shares 89% and 79% aa sequence identity with human and mouse TNF-, 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-and TNF-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.

This assay 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 coloured 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.

Figure 1:Schematic diagram of the assay

1. Aluminium pouches with a Microwell Plate coated with antibody to swine TNF-α(812)

2. 2 vials swine TNF-α Standard lyophilized, 2000 pg/ml upon reconstitution

3. 2 vials concentrated Biotin-Conjugate anti-swine TNF-α antibody

4. 2 vials Streptavidin-HRP solution,

5. 1 bottle Standard /sample Diluent

6. 1 bottle Biotin-Conjugate antibody Diluent

7. 1 bottle Streptavidin-HRP Diluent

8. 1 bottle Wash Buffer Concentrate 20x (PBS with 1% Tween-20)

9. 1 vial Substrate Solution

10. 1 vial Stop Solution

11. 4 pieces Adhesive Films

12. package insert

NOTE: [96 Tests]

Unopened Kit：Store at 2 -8° C. Do not use past kit expiration date. opened/ReconstitutedReagents：Please refer to the datasheets for detail information.

1. Trebichavsky, I. et al. (1995) Folia Microbiol. 40:417.

2. Allen, D.G. et al. (2001) Toxicol. Lett. 119:209.

3. Newman, W.H. et al. (1998) J. Surg. Res. 80:129.

4. Vezina, S-A. et al. (1995) Clin. Diag. Lab. Immunol. 2:665.

5. Pauli, U. (1995) Vet. Immunol. Immunopathol. 47:187.

6. Pauli, U. et al. (1989) Gene 81:185.

7. Von Niederhausen, B. et al. (1993) Vet. Immunol. Immunopathol. 38:57.

8. Kuhnert, P. et al. (1991) Gene 102:171.

9. Kwon, B. et al. (1999) Curr. Opin. Immunol. 11:340.

10. Idriss, H.T. and J.H. Naismith (2000) Microsc. Res. Tech. 50:184.

11. Sedgwick, J.D. et al. (2000) Immunol. Today 21:110.

12. Thomas, P.S. (2001) Immunol. Cell Biol. 79:132.

13. Saltiel, A.R. et al. (2001) Cell 104:517.

14. D’Haens, G. (2003) Curr. Pharm. Des. 9:289.

15. Feldmann, M. and R.N. Maini (2001) Annu. Rev. Immunol. 19:163.