Feline TNF-alpha DuoSet ELISA Summary
* 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 feline 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
Product Datasheets
Preparation and Storage
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.
Assay Procedure
GENERAL ELISA PROTOCOL
Plate Preparation
- 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.
- 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.
- Block plates by adding 300 μL Reagent Diluent to each well. Incubate at room temperature for a minimum of 1 hour.
- Repeat the aspiration/wash as in step 2. The plates are now ready for sample addition.
Assay Procedure
- 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.
- Repeat the aspiration/wash as in step 2 of Plate Preparation.
- 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.
- Repeat the aspiration/wash as in step 2 of Plate Preparation.
- 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.
- Repeat the aspiration/wash as in step 2.
- Add 100 μL of Substrate Solution to each well. Incubate for 20 minutes at room temperature. Avoid placing the plate in direct light.
- Add 50 μL of Stop Solution to each well. Gently tap the plate to ensure thorough mixing.
- 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 Feline 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.
7
Citations: Showing 1 - 7
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Clinical and pathological findings in fatal cases of severe fever with thrombocytopenia syndrome with high viremia in cats
Authors: A Matsuu, H Hatai, T Hifumi, E Hamakubo, M Take, T Tanaka, Y Momoi, Y Endo, A Koyoshi, Y Kamikubo, R Kamatsuki, N Kurusu, H Tabata, H Matsuyama, M Yanaidani, M Kawabata, T Kawabata
Topics in companion animal medicine, 2022-12-28;52(0):100756.
Species: Feline
Sample Types: Serum
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Clinical Relevance of the serum CTLA-4 in Cats with Mammary Carcinoma
Authors: AC Urbano, C Nascimento, M Soares, J Correia, F Ferreira
Sci Rep, 2020-03-02;10(1):3822.
Species: Feline
Sample Types: Serum
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Adipokines secretion in feline primary adipose tissue culture in response to dietary fatty acids
Authors: M Mazaki-Tov, SR Bolin, PA Schenck
BMC Vet. Res., 2019-09-06;15(1):324.
Species: Feline
Sample Types: Cell Culture Supernates
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Human and feline adipose-derived mesenchymal stem cells have comparable phenotype, immunomodulatory functions, and transcriptome
Authors: KC Clark, FA Fierro, EM Ko, NJ Walker, B Arzi, CG Tepper, H Dahlenburg, A Cicchetto, A Kol, L Marsh, WJ Murphy, N Fazel, DL Borjesson
Stem Cell Res Ther, 2017-03-20;8(1):69.
Species: Feline
Sample Types: Cell Culture Supernates
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INDUCTION OF CYTOKINE PRODUCTION IN CHEETAH (ACINONYX JUBATUS) PERIPHERAL BLOOD MONONUCLEAR CELLS AND VALIDATION OF FELINE-SPECIFIC CYTOKINE ASSAYS FOR ANALYSIS OF CHEETAH SERUM.
Authors: Franklin A, Crosier A, Vansandt L, Mattson E, Xiao Z
J Zoo Wildl Med, 2015-06-01;46(2):306-13.
Species: Feline - Cheetah
Sample Types: Cell Culture Supernates
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Failure of a dietary model to affect markers of inflammation in domestic cats.
Authors: Verbrugghe A, Janssens G, Van de Velde H, Cox E, De Smet S, Vlaeminck B, Hesta M
BMC Vet Res, 2014-05-04;10(0):104.
Species: Feline
Sample Types: Serum
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Activation of p38 MAPK by feline infectious peritonitis virus regulates pro-inflammatory cytokine production in primary blood-derived feline mononuclear cells.
Authors: Regan AD, Cohen RD, Whittaker GR
Virology, 2008-12-05;384(1):135-43.
Species: Feline
Sample Types: Cell Culture Supernates
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Percent recovery in serum improves as dilution increases. A 1:2 dilution results in approximately 45% recovery.
