Canine TNF-alpha DuoSet ELISA

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Ancillary Products Available
Canine TNF-alpha ELISA Standard Curve
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Product Details
Citations (15)
Supplemental Products

Canine TNF-alpha DuoSet ELISA Summary

Assay Type
Solid Phase Sandwich ELISA
96-well strip plate
Sample Volume Required
100 µL
Assay Range
15.6 - 1,000 pg/mL
Sufficient Materials
For fifteen 96-well plates*
Please see the product datasheet

* Provided that the recommended microplates, buffers, diluents, substrates and solutions are used, and the assay is run as summarized in the Assay Procedure provided.

This DuoSet ELISA Development kit contains the basic components required for the development of sandwich ELISAs to measure natural and recombinant canine TNF-alpha. The suggested diluent is suitable for the analysis of most cell culture supernate samples. Diluents for complex matrices, such as serum and plasma, should be evaluated prior to use in this DuoSet.

Product Features

  • Optimized capture and detection antibody pairings with recommended concentrations save lengthy development time
  • Development protocols are provided to guide further assay optimization
  • Assay can be customized to your specific needs
  • Economical alternative to complete kits

Kit Content

  • Capture Antibody
  • Detection Antibody
  • Recombinant Standard
  • Streptavidin conjugated to horseradish-peroxidase (Streptavidin-HRP)

Other Reagents Required

DuoSet Ancillary Reagent Kit 2 (5 plates): (Catalog # DY008) containing 96 well microplates, plate sealers, substrate solution, stop solution, plate coating buffer (PBS), wash buffer, and Reagent Diluent Concentrate 2.

The components listed above may be purchased separately:

PBS: (Catalog # DY006), or 137 mM NaCl, 2.7 mM KCl, 8.1 mM Na2HPO4, 1.5 mM KH2PO4, pH 7.2 - 7.4, 0.2 µm filtered

Wash Buffer: (Catalog # WA126), or 0.05% Tween® 20 in PBS, pH 7.2-7.4

Reagent Diluent: (Catalog # DY995), or 1% BSA in PBS, pH 7.2-7.4, 0.2 µm filtered

Substrate Solution: 1:1 mixture of Color Reagent A (H2O2) and Color Reagent B (Tetramethylbenzidine) (Catalog # DY999)

Stop Solution: 2 N H2SO4 (Catalog # DY994)

Microplates: R&D Systems (Catalog # DY990)

Plate Sealers: ELISA Plate Sealers (Catalog # DY992)

Scientific Data

Canine TNF-alpha ELISA Standard Curve

Product Datasheets

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Preparation and Storage

The product is shipped at ambient temperature. Upon receipt, store it immediately at the temperature recommended below.
Stability & 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); 102139631 (Cynomolgus Monkey); 100033834 (Equine); 493755 (Feline); 100009088 (Rabbit)
Alternate Names:
APC1 protein; Cachectin; Cachetin; DIF; TNF; TNF, monocyte-derived; TNFA; TNF-A; TNFalpha; TNF-alpha; TNF-alphacachectin; TNFATNF, macrophage-derived; TNFG1F; 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

Assay Procedure


Plate Preparation

  1. Dilute the Capture Antibody to the working concentration in PBS without carrier protein. Immediately coat a 96-well microplate with 100 μL per well of the diluted Capture Antibody. Seal the plate and incubate overnight at room temperature.
  2. Aspirate each well and wash with Wash Buffer, repeating the process two times for a total of three washes. Wash by filling each well with Wash Buffer (400 μL) using a squirt bottle, manifold dispenser, or autowasher. Complete removal of liquid at each step is essential for good performance. After the last wash, remove any remaining Wash Buffer by aspirating or by inverting the plate and blotting it against clean paper towels.
  3. Block plates by adding 300 μL Reagent Diluent to each well. Incubate at room temperature for a minimum of 1 hour.
  4. Repeat the aspiration/wash as in step 2. The plates are now ready for sample addition.

Assay Procedure

  1. Add 100 μL of sample or standards in Reagent Diluent, or an appropriate diluent, per well. Cover with an adhesive strip and incubate 2 hours at room temperature.
  2. Repeat the aspiration/wash as in step 2 of Plate Preparation.
  3. Add 100 μL of the Detection Antibody, diluted in Reagent Diluent, to each well. Cover with a new adhesive strip and incubate 2 hours at room temperature.
  4. Repeat the aspiration/wash as in step 2 of Plate Preparation.
  5. Add 100 μL of the working dilution of Streptavidin-HRP to each well. Cover the plate and incubate for 20 minutes at room temperature. Avoid placing the plate in direct light.
  6. Repeat the aspiration/wash as in step 2.
  7. Add 100 μL of Substrate Solution to each well. Incubate for 20 minutes at room temperature. Avoid placing the plate in direct light.
  8. Add 50 μL of Stop Solution to each well. Gently tap the plate to ensure thorough mixing.
  9. Determine the optical density of each well immediately, using a microplate reader set to 450 nm. If wavelength correction is available, set to 540 nm or 570 nm. If wavelength correction is not available, subtract readings at 540 nm or 570 nm from the readings at 450 nm. This subtraction will correct for optical imperfections in the plate. Readings made directly at 450 nm without correction may be higher and less accurate.

Citations for Canine TNF-alpha DuoSet ELISA

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.

15 Citations: Showing 1 - 10
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  1. miR-148a regulation interferes in inflammatory cytokine and parasitic load in canine leishmaniasis
    Authors: GT Rebech, JP Bragato, SF Costa, JH de Freitas, MO Dos Santos, MF Soares, FR Eugênio, PSP Dos Santos, VMF de Lima
    PloS Neglected Tropical Diseases, 2023-01-31;17(1):e0011039.
    Species: Canine
    Sample Types: Cell Culture Supernates
  2. miRNA-21 regulates CD69 and IL-10 expression in canine leishmaniasis
    Authors: JP Bragato, GT Rebech, JH Freitas, MOD Santos, SF Costa, FR Eugênio, PSPD Santos, VMF de Lima
    PLoS ONE, 2022-03-24;17(3):e0265192.
    Species: Canine
    Sample Types: cell culture supernatant
  3. Fluctuations in quality of life and immune responses during intravenous immunoglobulin infusion cycles
    Authors: JK Abbott, SK Chan, M MacBeth, JL Crooks, C Hancock, V Knight, EW Gelfand
    PLoS ONE, 2022-03-22;17(3):e0265852.
    Species: Canine
    Sample Types: cell culture supernatant
  4. Comparison of circulating CD4+, CD8+ lymphocytes and cytokine profiles between dogs with atopic dermatitis and healthy dogs
    Authors: MT Verde, S Villanueva, A Loste, D Marteles, D Pereboom, T Conde, A Fernández
    Research in Veterinary Science, 2022-01-29;145(0):13-20.
    Species: Canine
    Sample Types: Serum
  5. Placenta-derived multipotent mesenchymal stromal cells: a promising potential cell-based therapy for canine inflammatory brain disease
    Authors: RM Amorim, KC Clark, NJ Walker, P Kumar, K Herout, DL Borjesson, A Wang
    Stem Cell Res Ther, 2020-07-22;11(1):304.
    Species: Canine
    Sample Types: Cell Culture Supernates
  6. Local immune and microbiological responses to mucosal administration of a Liposome-TLR agonist immunotherapeutic in dogs
    Authors: W Wheat, L Chow, A Kuzmik, S Soontarara, J Kurihara, M Lappin, S Dow
    BMC Vet. Res., 2019-09-13;15(1):330.
    Species: Canine
    Sample Types: Cell Culture Supernates
  7. Effects of irradiation and leukoreduction on down regulation of CXCL-8 and storage lesion in stored canine whole blood
    Authors: H Yang, W Kim, J Bae, H Kim, S Kim, J Choi, J Park, DI Jung, H Koh, D Yu
    J. Vet. Sci., 2019-01-31;0(0):.
    Species: Canine
    Sample Types: Plasma
  8. PD-1 and PD-L1 regulate cellular immunity in canine visceral leishmaniasis
    Authors: KL Oliveira S, V Marin Chik, G Luvizotto, AA Correa Lea, BF de Almeida, F De Rezende, PSP Dos Santos, G Fabrino Ma, VMF De Lima
    Comp. Immunol. Microbiol. Infect. Dis., 2018-12-14;62(0):76-87.
    Species: Canine
    Sample Types: Cell Culture Supernates
  9. Early activation of deleterious molecular pathways in the kidney in experimental heart failure with atrial remodeling
    Authors: T Ichiki, BK Huntley, GJ Harty, SJ Sangaralin, JC Burnett
    Physiol Rep, 2017-05-15;5(9):.
    Species: Canine
    Sample Types: Tissue Homogenates
  10. Suppression of Canine Dendritic Cell Activation/Maturation and Inflammatory Cytokine Release by Mesenchymal Stem Cells Occurs Via Multiple Distinct Biochemical Pathways
    Stem Cells Dev., 2016-12-22;0(0):.
    Species: Canine
    Sample Types: Cell Culture Supernates
  11. Impact of LbSapSal Vaccine in Canine Immunological and Parasitological Features before and after Leishmania chagasi-Challenge
    PLoS ONE, 2016-08-24;11(8):e0161169.
    Species: Canine
    Sample Types: Cell Culture Supernates
  12. Targeted Doxorubicin Delivery to Brain Tumors via Minicells: Proof of Principle Using Dogs with Spontaneously Occurring Tumors as a Model
    Authors: JA MacDiarmid, V Langova, D Bailey, ST Pattison, SL Pattison, N Christense, LR Armstrong, VN Brahmbhatt, K Smolarczyk, MT Harrison, M Costa, NB Mugridge, I Sedliarou, NA Grimes, DL Kiss, B Stillman, CL Hann, GL Gallia, RM Graham, H Brahmbhatt
    PLoS ONE, 2016-04-06;11(4):e0151832.
    Species: Canine
    Sample Types: Serum
  13. Evaluation of Live Recombinant Nonpathogenic Leishmania tarentolae Expressing Cysteine Proteinase and A2 Genes as a Candidate Vaccine against Experimental Canine Visceral Leishmaniasis.
    Authors: Shahbazi M, Zahedifard F, Taheri T, Taslimi Y, Jamshidi S, Shirian S, Mahdavi N, Hassankhani M, Daneshbod Y, Zarkesh-Esfahani S, Papadopoulou B, Rafati S
    PLoS ONE, 2015-07-21;10(7):e0132794.
    Species: Canine
    Sample Types: Cell Culture Supernates
  14. One-year timeline kinetics of cytokine-mediated cellular immunity in dogs vaccinated against visceral leishmaniasis.
    Authors: Costa-Pereira C, Moreira M, Soares R, Marteleto B, Ribeiro V, Franca-Dias M, Cardoso L, Viana K, Giunchetti R, Martins-Filho O, Araujo M
    BMC Vet Res, 2015-04-11;11(0):92.
    Species: Canine
    Sample Types: Cell Culture Supernates
  15. Expression of regulatory T cells in jejunum, colon, and cervical and mesenteric lymph nodes of dogs naturally infected with Leishmania infantum.
    Authors: Figueiredo M, Deoti B, Amorim I, Pinto A, Moraes A, Carvalho C, da Silva S, de Assis A, de Faria A, Tafuri W
    Infect Immun, 2014-06-16;82(9):3704-12.
    Species: Canine
    Sample Types: Tissue Homogenates


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