Sheep Tumor Necrosis Factor Alpha (TNFa) ELISA Kit

Principle of the Assay

The microtiter plate provided in this kit has been pre-coated with an antibody specific to TNFa. Standards or samples are then added to the appropriate microtiter plate wells with a biotin-conjugated antibody preparation specific to TNFa. Next, Avidin conjugated to Horseradish Peroxidase (HRP) is added to each microplate well and incubated. After the TMB substrate solution is added, only those wells that contain TNFa, biotin-conjugated antibody, and enzyme-conjugated Avidin will exhibit a change in color. The enzyme-substrate reaction is terminated by the addition of sulphuric acid solution, and the color change is measured spectrophotometrically at a wavelength of 450nm ± 10nm. The concentration of TNFa in the samples is then determined by comparing the O.D. of the samples to the standard curve.

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

Target Information

Cytokine that binds to TNFRSF1A/TNFR1 and TNFRSF1B/TNFBR. It is mainly secreted by macrophages and can induce cell death of certain tumor cell lines. It is potent pyrogen causing fever by direct action or by stimulation of interleukin-1 secretion and is implicated in the induction of cachexia, Under certain conditions it can stimulate cell proliferation and induce cell differentiation (By similarity). Induces insulin resistance in adipocytes via inhibition of insulin-induced IRS1 tyrosine phosphorylation and insulin-induced glucose uptake. Induces GKAP42 protein degradation in adipocytes which is partially responsible for TNF-induced insulin resistance (By similarity). Plays a role in angiogenesis by inducing VEGF production synergistically with IL1B and IL6 (By similarity).

GENE ID	443540
SWISS PROT	P23383
SYNONYMS	DIF; TNF-A; TNFSF2; Cachectin; Tumor Necrosis Factor Ligand Superfamily Member 2

Materials Supplied

Kit Components	96 Wells Quantity/Size
Pre-coated, ready-to-use 96-well strip plate	1 plate
Plate sealer for 96 wells	2
Standard	2 tubes
Diluent buffer	1 bottle
Detection Reagent A	1 bottle
Detection Reagent B	1 bottle
TMB Substrate	1 tube
Stop Solution	1 tube
Wash Buffer (30 ℅ concentrate)	1 tube
Product data sheet	1 copy

Storage

The TMB Substrate, Wash Buffer (30X concentrate), and the Stop Solution should be stored at 4°C upon receipt, while the other items should be stored at -20°C.

Performance Characteristics

REPEATABILITY	Intra-assay Precision (Precision within an assay): 3 samples with low, middle, and high-level TNFa were tested 20 times on one plate, respectively. Inter-assay Precision (Precision between assays): 3 samples with low, middle, and high-level TNFa were tested on 3 different plates, with 8 replicates in each plate. CV(%) = SD/meanX100 Intra-Assay: CV<10% Inter-Assay: CV<12%
SENSITIVITY	The minimum detectable dose was 7.1pg/mL.
ASSAY RANGE	15.6-1000pg/mL
SPECIFICITY	This assay has high sensitivity and excellent specificity for the detection of TNFa. No significant cross-reactivity or interference between TNFa and analogs was observed. Note: Limited by current skills and knowledge, it is impossible to perform all possible cross-reactivity detection tests between TNFa and all analogs, therefore, cross reactivity may still exist.