Swine 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. 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. It is a pleiotropic molecule that plays a central role in inflammation, apoptosis, and immune system development. TNF-α is produced by a wide variety of immune and epithelial cell types. Human TNF-α consists of a 35 amino acid (aa) cytoplasmic domain, a 21 aa transmembrane segment, and a 177 aa extracellular domain (ECD). Within the ECD, human TNF-α shares 97% aa sequence identity with rhesus and 71% - 92% with bovine, canine, cotton rat, equine, feline, mouse, porcine, and rat TNF-α. The 26 kDa type 2 transmembrane protein is assembled intracellularly to form a noncovalently linked homotrimer. Ligation of this complex induces reverse signaling that promotes lymphocyte costimulation but diminishes monocyte responsiveness.

Cleavage of membrane bound TNF-α by TACE/ADAM17 releases a 55 kDa soluble trimeric form of TNF-α. TNF-α trimers bind the ubiquitous TNF RI and the hematopoietic cell-restricted TNF RII, both of which are also expressed as homotrimers. TNF-α regulates lymphoid tissue development through control of apoptosis. It also promotes inflammatory responses by inducing the activation of vascular endothelial cells and macrophages. TNF-α is a key cytokine in the development of several inflammatory disorders. It contributes to the development of type 2 diabetes through its effects on insulin resistance and fatty acid metabolism.

GENE ID 397086
SWISS PROT P23563
SYNONYMS TNFa

Materials Supplied

Kit Components 96 Wells Quantity/Size
Aluminium pouches with a Microwell Plate coated with antibody to Swine TNF-α (8-12) 1 plate
Swine TNF-α standard lyophilized, 2000pg/ml upon reconstitution 2 vials
Concentrated Biotin-Conjugate anti-Swine 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

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 Swine 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, s TNF RII/Fc
Recombinant mouse TNF-α(truncated, s TNF RI, s TNF RII IL-4