Mouse Tumor necrosis factor α (TNF-α) ELISA Kit

This assay employs a two-site sandwich ELISA to quantitate TNF-α in samples. An 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. After removing any unbound substances, a biotin-conjugated antibody specific for TNF-α is added to the wells. After washing, Streptavidin conjugated Horseradish Peroxidase (HRP) is added to the wells. Following a wash to remove any unbound avidin-enzyme reagent, a substrate solution is added to the wells and color develops in proportion to the amount of TNF-α bound in the initial step. The color development is stopped and the intensity of the color is measured.

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

This assay has high sensitivity and excellent specificity for detection of Mouse TNF-α. No significant cross-reactivity or interference between Mouse TNF-α and analogues was observed.

Matrices listed below were spiked with certain level of recombinant Mouse TNF-α and the recovery rates were calculated by comparing the measured value to the expected amount of Mouse TNF-α in samples.

The linearity of the kit was assayed by testing samples spiked with appropriate concentration of Mouse TNF-α and their serial dilutions. The results were demonstrated by the percentage of calculated concentration to the expected.