Human TNF-Alpha ELISA Assay


The Eagle Biosciences Human TNF Alpha ELISA Assay Kit (enzyme-linked immunoassay kit) is intended for the quantitative determination of human TNF Alpha (TNF-α) concentrations in cell culture supernates, serum, and plasma. The Eagle Biosciences Human TNF Alpha ELISA Assay Kit is for research use only and not to be used in diagnostic procedures.

SKU: TNA31-K01 Categories: , ,

Human TNF-Alpha ELISA Assay

For Research Use Only

Size: 1×96 wells
Sensitivity: 7 pg/mL
Dynamic Range: 31.25 – 1000 pg/ml
Incubation Time: 3.5 hours
Sample Type: Serum, Plasma, Cell Culture
Sample Size: 100 µl

Additional Information

Assay Background

The prototype ligand of the TNF superfamily, TNF-α/TNFSF1A, is a pleiotropic cytokine that plays a central role in inflammation and apoptosis (1-4). Human cells known to express TNF-α include B cells, colonic columnar epithelial cells, NK and CD3+CD56+ hepatic natural T cells, macrophages, monocytes and monocyte-derived dendritic cells, CD4+ and CD8+ T cells, mast cells, neutrophils, keratinocytes, plasma cells, and adipocytes.

It is synthesized as a 26 kDa, type II transmembrane protein that is 233 amino acids (aa) in length (4, 5). It contains a 30 aa cytoplasmic domain, a 26 aa transmembrane segment, and a 177 aa extracellular region (6, 7). TNF-α is assembled intracellularly to form a transmembrane, non-covalently-linked homotrimeric protein. The 157 aa residue soluble form of TNF-α (sTNF-α is released from the C-terminus of the transmembrane protein through the activity of TNF-α-converting enzyme (TACE), a membrane-bound disintegrin metalloproteinase (8, 9).

TNF-α is reported to promote inflammatory cell infiltration by upregulating leukocyte adhesion molecules on endothelial cells, serve as a chemotactic agent for monocytes, and activate phagocyte killing mechanisms (10). Deficiencies in either TNF-α or its receptors can increase susceptibility to infection by intracellular pathogens (11 – 12). TNF- may also play a role in lymphoid tissue development. Knockout mice lack splenic B cell follicles and the ability to form germinal centers (13, 14). Other potential physiological roles for TNF-α and its receptors include regulating the differentiation of hematopoietic stem and progenitor cells (15 – 17).  TNF-α has been implicated in a number of pathophysiological processes. It is associated with unregulated pro-inflammatory activity and is thought to be a critical mediator of endotoxin-induced septic shock (18). Cachexia (or whole body wasting) has also been associated with long-term circulating TNF-α.

Assay Principle

The Eagle Biosciences Human TNF 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. Standards and samples are pipetted into the wells and any TNF-α present is bound by the immobilized antibody. Following incubation unbound samples are removed during a wash step, and then a detection antibody specific for TNF-α is added to the wells and 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.

Assay Procedure

  1. Prepare all reagents and working standards as directed in the previous sections.
  2. 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.
  3. Add 100 µL of Standard, control, or sample, per well. Cover with the adhesive strip provided. Incubate for 1.5 hours at 37C.
  4. 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.
  5. Add 100 µL of the working solution of Biotin-Conjugate to each well. Cover with a new adhesive strip and incubate 1 hour at 37C.
  6. Repeat the aspiration/wash.
  7. 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 37C.  Avoid placing the plate in direct light.
  8. Repeat the aspiration/wash.
  9. Add 100 µL of Substrate Solution to each well. Incubate for 10-20 minutes at 37C. Avoid placing the plate in direct light.
  10. Add 100 µL of Stop Solution to each well. Gently tap the plate to ensure thorough mixing.
  11. 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)


Product Manual



  1. Kwon, B. et al. (1999) Curr. Opin. Immunol. 11:340.
  2. Idriss, H.T. and J.H. Naismith (2000) Microsc. Res. Tech. 50:184.
  3. Sedgwick, J.D. et al. (2000) Immunol. Today 21:110.
  4. MacEwan, D.J. (2002) Brit. J. Pharmacol. 135:855.
  5. Pennica, D. et al. (1984) Nature 312:724.
  6. Wang, A.M. et al. (1985) Science 228:149.
  7. Ishisaka, R. et al. (1999) J. Biochem. 126:413.
  8. Kriegler, M. et al. (1988) Cell 53:45.
  9. Moss, M.L. et al. (1997) Nature 385:733.
  10. Wang, C.Y. et al. (1998) Science 281:1680.
  11. Peschon, J.J. et al. (1998) J. Immunol. 160:943.
  12. Wellmer, A. et al. (2001) Infect. Immun. 69:6881.
  13. Trevejo, J.M. et al. (2001) Proc. Natl. Acad. Sci. USA 98:12162.
  14. Kasahara, S. et al. (2003) J. Virol. 77:2469.
  15. Pasparakis, M. et al. (1996) J. Exp. Med. 184:1397.
  16. Taniguchi, T. et al. (1997) Lab. Invest. 77:647.
  17. Jacobsen, F.W. et al. (1994) Proc. Natl. Acad. Sci. USA 91:10695.
  18. Zhang, Y. et al. (1995) Blood 86:2930.