Human 4-1BB/TNFRSF9 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 human 4-1BB/TNFRSF9. 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: 4-1BB/TNFRSF9/CD137
4-1BB, also known as CD137 and TNFRSF9, is a transmembrane TNF receptor superfamily member that is expressed as on various populations of activated T cell including CD4+, CD8+, memory CD8+, NKT, and regulatory T cells as well as on myeloid and mast cell progenitors, dendritic cells, mast cells, and bacterially infected osteoblasts. 4-1BB binding by 4-1BB Ligand costimulates T cell proliferation, activation, and/or survival and can enhance the activation-induced cell death of repetitively stimulated T cells. 4-1BB can associate with OX40 on activated T cells, forming a complex that responds to either ligand and inhibits Treg and CD8+ T cell proliferation. Reverse signaling through 4-1BB Ligand inhibits the development of dendritic cells, B cells, and osteoclasts but supports mature dendritic cell survival and costimulates the proliferation and activation of mast cells.
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 Human 4-1BB/TNFRSF9 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.
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Citations: Showing 1 - 7
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Soluble CD137: A Potential Prognostic Biomarker in Critically Ill Patients
Authors: Räth, U;Mester, P;Schwarz, H;Schmid, S;Müller, M;Buechler, C;Pavel, V;
International journal of molecular sciences
Species: Human
Sample Types: Plasma
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Soluble CD137 is a novel serum marker of liver cirrhosis in patients with hepatitis C and alcohol-associated disease etiology
Authors: K Weigand, G Peschel, J Grimm, K Luu, D Schacherer, R Wiest, M Müller, H Schwarz, C Buechler
European Journal of Immunology, 2021-12-26;0(0):.
Species: Human
Sample Types: Serum
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Clinical significance of the TNF-? receptors, TNFRSF2 and TNFRSF9, on cell migration molecules Fascin-1 and Versican in acute leukemia
Authors: N El Kramani, NM Elsherbiny, AM El-Gayar, MA Ebrahim, MMH Al-Gayyar
Cytokine, 2018-06-01;0(0):.
Species: Human
Sample Types: Plasma
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Levels of 4-1BB transcripts and soluble 4-1BB protein are elevated in the adipose tissue of human obese subjects and are associated with inflammatory and metabolic parameters.
Authors: Tu T, Kim C, Kang J, Nam-Goong I, Nam C, Kim E, Kim Y, Choi J, Kawada T, Goto T, Park T, Yoon Park J, Choi M, Yu R
Int J Obes (Lond), 2013-11-27;38(8):1075-82.
Species: Human
Sample Types: Tissue Homogenates
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Decreased 4-1BB expression on CD4+CD25 high regulatory T cells in peripheral blood of patients with multiple sclerosis.
Authors: Liu GZ, Gomes AC, Fang LB, Gao XG, Hjelmstrom P
Clin. Exp. Immunol., 2008-08-22;154(1):22-9.
Species: Human
Sample Types: Plasma
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Potential novel biomarkers of disease activity in rheumatoid arthritis patients: CXCL13, CCL23, transforming growth factor alpha, tumor necrosis factor receptor superfamily member 9, and macrophage colony-stimulating factor.
Authors: Rioja I, Hughes FJ, Sharp CH, Warnock LC, Montgomery DS, Akil M, Wilson AG, Binks MH, Dickson MC
Arthritis Rheum., 2008-08-01;58(8):2257-67.
Species: Human
Sample Types: Plasma
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CD137 is expressed in human atherosclerosis and promotes development of plaque inflammation in hypercholesterolemic mice.
Authors: Olofsson PS, Soderstrom LA, Wagsater D, Sheikine Y, Ocaya P, Lang F, Rabu C, Chen L, Rudling M, Aukrust P, Hedin U, Paulsson-Berne G, Sirsjo A, Hansson GK
Circulation, 2008-02-19;117(10):1292-301.
Species: Human
Sample Types: Tissue Homogenates
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