Human TNF-α ELISA Kit

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. 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 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

The prototype ligand of the TNF superfamily, TNF-α/TNFSF1A, is a pleiotropic cytokine that plays a central role in inflammation and apoptosis. 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. It contains a 30 aa cytoplasmic domain, a 26 amino acids transmembrane segment, and a 177 amino acids extracellular region. 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).

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. Deficiencies in either TNF-α or its receptors can increase susceptibility to infection by intracellular pathogens. TNF- may also play a role in lymphoid tissue development. Knockout mice lack splenic B cell follicles and the ability to form germinal centers. Other potential physiological roles for TNF-α and its receptors include regulating the differentiation of hematopoietic stem and progenitor cells.

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. Cachexia (or whole body wasting) has also been associated with long-term circulating TNF-α. Other disorders with potential TNF-α involvement include asthma), type 2 diabetes, Crohn’s disease, and rheumatoid arthritis.

GENE ID 7124
SWISS PROT P01375
SYNONYMS DIF-alpha, TNFA, TNFSF2, TNLG1F, TNF

Materials Supplied

Kit Components 96 Wells Quantity/Size
Aluminium pouches with a Microwell Plate coated with monoclonal antibody to human TNF-α(8﹡12) 1 plate
Human TNF-α Standard lyophilized, 4000 pg/ml upon reconstitution 2 vials
Concentrated Biotin-Conjugate anti-human TNF-αmonoclonal 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

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 7pg/mL.
ASSAY RANGE 15.6 - 1000 pg/mL
SPECIFICITY This assay recognizes both natural and recombinant human TNF-α. The factors listed below were prepared at 70ng/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 human TNF-β, GM-CSF, s TNF RI, s TNF RII/Fc, s TNF RII
Recombinant mouse TNF-α, TNF-α(truncayed), s TNF RI, s TNF RII IL-4
Recombinant rat TNF-α