Bovine TNF-alpha DuoSet ELISA

Catalog # Availability Size / Price Qty
DY2279
Ancillary Products Available
Bovine TNF-alpha ELISA Standard Curve
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Product Details
Procedure
Citations (8)
FAQs
Supplemental Products
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Bovine TNF-alpha DuoSet ELISA Summary

Assay Type
Solid Phase Sandwich ELISA
Format
96-well strip plate
Sample Volume Required
100 µL
Sufficient Materials
For fifteen 96-well plates*
Specificity
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 bovine 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 3 (5 plates): (Catalog # DY009) containing 96 well microplates, plate sealers, substrate solution, stop solution, plate coating buffer (PBS), wash buffer, and Reagent Diluent Concentrate 3.

Normal Goat Serum: (Catalog # DY005)

 

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 # DY004), or 5% Tween 20 in PBS, 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)

Normal Goat Serum: (Catalog # DY005)

 

Data Example

Bovine TNF-alpha ELISA Standard Curve

Product Datasheets

Preparation and Storage

Shipping
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); 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

GENERAL ELISA PROTOCOL


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 of 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 with NGS, 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 Bovine 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.

8 Citations: Showing 1 - 8
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  1. LP induced/mediated PGE2 synthesis through activation of the ERK/NF-?B pathway contributes to inflammatory damage triggered by Escherichia coli-infection in bovine endometrial tissue
    Authors: T Li, W Mao, B Liu, R Gao, S Zhang, J Wu, C Fu, Y Deng, K Liu, Y Shen, J Cao
    Vet. Microbiol., 2019;232(0):96-104.
    Species: Bovine
    Sample Types: Tissue Culture Supernates
  2. Lipid-Rich Extract from Mexican Avocado Seed (Persea americana var. drymifolia) Reduces Staphylococcus aureus Internalization and Regulates Innate Immune Response in Bovine Mammary Epithelial Cells
    Authors: M Báez-Magañ, A Ochoa-Zarz, N Alva-Muril, R Salgado-Ga, JE López-Meza
    J Immunol Res, 2019;2019(0):7083491.
    Species: Bovine
    Sample Types: Cell Culture Supernates
  3. Characterization of the endocannabinoid system in subcutaneous adipose tissue in periparturient dairy cows and its association to metabolic profiles
    Authors: M Zachut, G Kra, U Moallem, L Livshitz, Y Levin, S Udi, A Nemirovski, J Tam
    PLoS ONE, 2018;13(11):e0205996.
    Species: Bovine
    Sample Types: Plasma
  4. Regulation of cytokine gene expression by orosomucoid in neonatal swine adipose tissue
    Authors: TG Ramsay, MJ Stoll, le A Blomberg, TJ Caperna
    J Anim Sci Biotechnol, 2016;7(0):25.
    Species: Bovine
    Sample Types: Cell Culture Supernates
  5. Nitric oxide modulates the immunological response of bovine PBMCs in an in vitro BRDc infection model
    Authors: Chris Miller
    Res. Vet. Sci., 2016;109(0):21-28.
    Species: Bovine
    Sample Types: Cell Culture Supernates
  6. Impact of in vitro treatments of physiological levels of estradiol and progesterone observed in pregnancy on bovine monocyte-derived dendritic cell differentiation and maturation
    Authors: Ynte Schukken
    Vet. Immunol. Immunopathol., 2016;182(0):37-42.
    Species: Bovine
    Sample Types: Cell Culture Supernates
  7. Epithelial and stromal cells of bovine endometrium have roles in innate immunity and initiate inflammatory responses to bacterial lipopeptides in vitro via Toll-like receptors TLR2, TLR1, and TLR6.
    Authors: Turner M, Cronin J, Healey G, Sheldon I
    Endocrinology, 2014;155(4):1453-65.
    Species: Bovine
    Sample Types: Cell Culture Supernates
  8. Plasma levels of TNF-alpha, IFN-gamma, IL-4 and IL-10 during a course of experimental contagious bovine pleuropneumonia.
    Authors: Sacchini F, Luciani M, Salini R, Scacchia M, Pini A, Lelli R, Naessens J, Poole J, Jores J
    BMC Vet. Res., 2012;8(0):44.
    Species: Bovine
    Sample Types: Plasma

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