Tumor necrosis factor alpha (TNF-α), also known as cachectin and TNFSF1A, is the prototype ligand of the tumor necrosis factor superfamily. TNF-α is a 26 kDa type II transmembrane protein consisting of a 35 amino acid (aa) intracellular domain, a 21 aa transmembrane segment, and a 177 aa extracellular domain (ECD). TNF-α signaling is mediated by two receptors: TNFR1 and TNFR2. TNFR1 is expressed on most cell types, whereas TNFR2 is restricted to endothelial, epithelial, and subsets of immune cells. TNF-α plays a central role in inflammatory response, immune system development, apoptosis, and lipid metabolism. TNF-α is also involved in many pathological processes, including asthma, Crohn's disease, rheumatoid arthritis, neuropathic pain, obesity, type 2 diabetes, septic shock, autoimmunity, and cancer.

This assay kit employs the double antibody sandwich enzyme-linked immunosorbent assay (ELISA) technique. Specific anti-mouse TNF-α antibodies with high affinity are pre-coated onto the enzymelinked plate. In the wells of the enzyme-linked plate, standards, test samples, and biotinylated detection antibodies are added. After incubation, TNF-α present in the samples binds to the solid-phase antibody and the detection antibody, forming an immune complex. After removing unbound substances, streptavidin-horseradish peroxidase labeled with horseradish peroxidase (HRP) is added. After wash step to remove any unbound substances, 3,3′,5,5′-Tetramethylbenzidine (TMB)substrate is added and color develops in proportion to the amount of TNF-α bound initially. The assay is stopped, and the optical density of the wells is determined using a micro-plate reader at 450nm wavelength (with a reference wavelength of 540nm or 570nm) for quantification.

Recovery
Mouse TNF-α of different levels was spiked into cell culture medium samples to determine its recovery rate. The recovery rate ranged from 88-107%, with an average recovery rate of 94%.
Sensitivity
The minimum detectable dose (MDD) of mouse TNF-α is typically less than 5.5 pg/mL.
The MDD was determined by adding two standard deviations to the mean O.D. value of twenty zero standard replicates and calculating the corresponding concentration.
Linearity
To assess the linearity of the assay, four samples spiked with high concentrations of mouse TNF-α were diluted with 1×Reagent Diluent to bring them within the detection range.

Specificity
This assay recognizes natural and recombinant mouse TNF-α.
The factors listed below were prepared at 50 ng/mL in 1×Reagent Diluent and assayed for cross-reactivity.
Preparations of the following factors prepared at 50 ng/mL in a mid-range mouse TNF-α control were assayed for interference. No significant cross-reactivity or interference was observed.