Principle of the Assay

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.

For Use with serum, plasma and cell culture supernatants. For Research Use Only. Not for use in diagnostic procedures.

Target Information

Tumor necrosis factor alpha (TNF-α), also known as cachectin and TNFSF2, is the prototypic ligand of the TNF superfamily. TNF-α is a mononuclear factor produced primarily by monocytes and macrophages. In 1975, Carswell et al. found that BCG attacked mice and then treated with endotoxin. A substance in the serum of mice that induced hemorrhagic necrosis of tumor tissue was named as tumor necrosis factor. In 1985, Shalaby named TNF produced by macrophages as TNF-α, and the lymphotoxin produced by T lymphocytes was named TNF-β.

Rat TNF-α is synthesized as a 26 kDa type II transmembrane protein that consists of a 35 amino acid (aa) cytoplasmic domain, a 21 aa transmembrane segment, and a 156 aa extracellular domain (ECD) (12). Within the ECD, rat TNF-α shares 95% aa sequence identity with mouse, and 73% - 79% aa identity with bovine, canine, cotton rat, equine, feline, human, rhesus macaque and porcine TNF-α. It is produced by a wide variety of immune, epithelial, endothelial and tumor cells.

The biological activities of TNF-α are very complex, including regulation of hematopoiesis, immunity and inflammation; effects on blood vessels and blood clotting. It also has the effects on various organs (liver, heart, bone, cartilage, muscle and other tissues). TNF-α regard as the growth factor for certain tumor cells. It has the synergy effect EGF, PDGF and insulin to promote the expression of EGF receptor. Recently, TNF-β (LT) has been reported to be an autocrine growth factor for Epstein-Barr virus-transformed lymphoblasts, and anti-LT antibodies, sTNF R. It also can inhibit the proliferation of Epstein-Barr virus-transformed lymphocytes.

The anti-tumor effects of TNF-α include the direct action of TNF-α and immune response that induced by TNF-α against tumors. TNF-α is involved in diseases including asthma, type II diabetes, Crohn's disease and rheumatoid arthritis. TNF stimulates endothelial cells that can cause inflammation, tissue damage and blood clotting to induce septic shock.

GENE ID 24835

Materials Supplied

Kit Components 96 Wells Quantity/Size
Aluminium pouches with a Microwell Plate coated with antibody to rat TNF-α (8-12) 1 plate
Rat TNF-α Standard lyophilized, 2000pg/ml upon reconstitution 2 vials
concentrated Biotin-Conjugate anti-rat TNF-α antibody 2 vials
Streptavidin-HRP solution 2 vials
Standard /sample Diluent 1 bottle
Biotin-Conjugate antibody Diluent 1 bottle
Streptavidin-HRP Diluent 1 bottle
Wash Buffer Concentrate 20x (PBS with 1% Tween-20) 1 bottle
Substrate Solution 1 vial
Stop Solution 1 vial
Adhesive Films 4 pieces
Product data sheet 1 copy


Storage Store at 2 - 8°C

Performance Characteristics

REPEATABILITY The coefficient of variation of both intra-assay and inter-assay were less than 10%.
SENSITIVITY The minimum detectable dose was 15pg/mL.
ASSAY RANGE 31.25 - 2000 pg/mL
SPECIFICITY This assay recognizes both natural and recombinant rat TNF-α. The factors listed below were prepared at 50ng/ml in Standard /sample Diluent and assayed for cross-reactivity and no significant cross-reactivity or interference was observed.
Factors assayed for cross-reactivity
Recombinant rat CINC-1, GDNF, β-NGF, PDGF-BB, IFN-γ, IL-1β, IL-2, IL-4
Recombinant human TNF-α, TNF-β, TNF sRI, TNF sRII
Recombinant mouse TNF sRI, TNF sRII