< 0.5% cross-reactivity observed with available related molecules.< 50% cross-species reactivity observed with species tested.
No significant interference observed with available related molecules.
The Quantikine Canine TNF-alpha Immunoassay is a 4.5 hour solid-phase ELISA designed to measure canine TNF-alpha in cell culture supernates, serum, and plasma. It contains E. coli-expressed recombinant canine TNF-alpha and antibodies raised against the recombinant protein. Results obtained for naturally occurring canine TNF-alpha showed linear curves that were parallel to the standard curves obtained using the Quantikine kit standards. These results indicate that this kit can be used to determine relative mass values for natural canine TNF-alpha.
Intra-Assay Precision (Precision within an assay) Three samples of known concentration were tested twenty times on one plate to assess intra-assay precision.
Inter-Assay Precision (Precision between assays) Three samples of known concentration were tested in separate assays to assess inter-assay precision.
The recovery of canine TNF-alpha spiked to levels throughout the range of the assay in various matrices was evaluated.
Average % Recovery
Cell Culture Supernates (n=7)
EDTA Plasma (n=6)
Heparin Plasma (n=6)
To assess the linearity of the assay, samples containing or spiked with high concentrations of canine TNF-alpha in each matrix were diluted with Calibrator Diluent and assayed.
Preparation and Storage
Store the unopened product at 2 - 8 °C. Do not use past expiration date.
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.
Refer to the product for complete assay procedure.
Bring all reagents and samples to room temperature before use. It is recommended that all samples, standards, and controls be assayed in duplicate.
Prepare all reagents, standard dilutions, and samples as directed in the product insert.
Remove excess microplate strips from the plate frame, return them to the foil pouch containing the desiccant pack, and reseal.
50 µL Assay Diluent
Add 50 µL of Assay Diluent to each well.
50 µL Standard, Control, or sample
Add 50 µL of Standard, Control, or sample to each well. Tap the plate gently for 1 minute. Cover with a plate sealer, and incubate at room temperature for 2 hours.
Aspirate each well and wash, repeating the process 4 times for a total of 5 washes.
100 µL Conjugate
Add 100 µL of Conjugate to each well. Cover with a new plate sealer, and incubate at room temperature for 1 hour.
Aspirate and wash 5 times.
100 µL Streptavidin-HRP
Add 100 µL Streptavidin-HRP to each well. Cover with a new plate sealer, and incubate at room temperature for 30 minutes.
Aspirate and wash 5 times.
100 µL Substrate Solution
Add 100 µL Substrate Solution to each well. Incubate at room temperature for 30 minutes. PROTECT FROM LIGHT.
100 µL Stop Solution
Add 100 µL of Stop Solution to each well. Gently tap the plate to ensure thorough mixing. Read at 450 nm within 30 minutes. Set wavelength correction to 540 nm or 570 nm.
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