Canine TNF-alpha Quantikine ELISA Kit

(3 citations)   
  • Assay Type
    Solid Phase Sandwich ELISA
  • Format
    96-well strip plate
  • Assay Length
    4.5 hours
  • Sample Type & Volume Required Per Well
    Cell Culture Supernates (50 uL), Serum (50 uL), EDTA Plasma (50 uL), Heparin Plasma (50 uL)
  • Sensitivity
    4.2 pg/mL
  • Assay Range
    7.8 - 500 pg/mL (Cell Culture Supernates, Serum, EDTA Plasma, Heparin Plasma)
  • Specificity
    Natural and recombinant canine TNF-alpha
  • Cross-reactivity
    < 0.5% cross-reactivity observed with available related molecules.< 50% cross-species reactivity observed with species tested.
  • Interference
    No significant interference observed with available related molecules.
Product Summary
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.
Cell Culture Supernates, Serum, EDTA Plasma, Heparin Plasma
Intra-Assay Precision Inter-Assay Precision
Standard Deviation1.56.515.


The recovery of canine TNF-alpha spiked to levels throughout the range of the assay in various matrices was evaluated.

Sample Type Average % Recovery Range %
Cell Culture Supernates (n=7) 99 83-109
EDTA Plasma (n=6) 94 82-103
Heparin Plasma (n=6) 94 88-103
Serum (n=6) 92 83-106
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.
Canine TNF-alpha Quantikine ELISA Kit
Preparation and Storage
  • Storage
    Store the unopened product at 2 - 8 °C. Do not use past expiration date.
Background: TNF-alpha

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.

    • Long Name
      Tumor Necrosis Factor alpha
    • Entrez Gene IDs
      7124 (Human); 21926 (Mouse); 24835 (Rat); 397086 (Porcine); 280943 (Bovine); 403922 (Canine); 100033834 (Equine); 493755 (Feline); 100009088 (Rabbit);
    • Alternate Names
      APC1 protein; Cachectin; Cachetin; DIF; TNF; TNF, monocyte-derived; tnfa; tnf-a; TNFalpha; TNF-alphacachectin; TNFATNF, macrophage-derived; TNFSF1A; TNFSF2; TNFSF2TNF superfamily, member 2; tumor necrosis factor (TNF superfamily, member 2); tumor necrosis factor alpha; Tumor necrosis factor ligand superfamily member 2; tumor necrosis factor; tumor necrosis factor-alpha;
    Related Research Areas
    Assay Procedure
    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.
    1.   Prepare all reagents, standard dilutions, and samples as directed in the product insert.
    2.   Remove excess microplate strips from the plate frame, return them to the foil pouch containing the desiccant pack, and reseal.

    3. 50 µL Assay Diluent
    4.   Add 50 µL of Assay Diluent to each well.

    5. 50 µL Standard, Control, or sample
    6.   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.
    7.   Aspirate each well and wash, repeating the process 4 times for a total of 5 washes.

    8. 100 µL Conjugate
    9.   Add 100 µL of Conjugate to each well. Cover with a new plate sealer, and incubate at room temperature for 1 hour.
    10.   Aspirate and wash 5 times.

    11. 100 µL Streptavidin-HRP
    12.   Add 100 µL Streptavidin-HRP to each well. Cover with a new plate sealer, and incubate at room temperature for 30 minutes.
    13.   Aspirate and wash 5 times.

    14. 100 µL Substrate Solution
    15. Add 100 µL Substrate Solution to each well. Incubate at room temperature for 30 minutes. PROTECT FROM LIGHT.

    16. 100 µL Stop Solution
    17. 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.

    R&D Systems personnel manually curate a database that contains references using R&D Systems products. The data collected includes not only links to publications in PubMed, but also provides information about sample types, species, and experimental conditions.

    Showing Results 1 - 3 of 3
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    Sample Type
    1. TLR5 risk-associated haplotype for canine inflammatory bowel disease confers hyper-responsiveness to flagellin.
      Authors: Kathrani A, Holder A, Catchpole B, Alvarez L, Simpson K, Werling D, Allenspach K
      PLoS ONE, 2012;7(1):e30117.
      Species: Canine
      Sample Type: Serum
    2. Human Flt3L generates dendritic cells from canine peripheral blood precursors: implications for a dog glioma clinical trial.
      Authors: Xiong W, Candolfi M, Liu C
      PLoS ONE, 2010;5(6):e11074.
      Species: Canine
      Sample Type: Cell Culture Supernates
    3. A functional comparison of canine and murine bone marrow derived cultured mast cells.
      Authors: Lin TY, London CA,
      Vet. Immunol. Immunopathol., 2006;114(3):320-34.
      Species: Canine
      Sample Type: Cell Culture Supernates
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