Mouse IL-12/IL-23 p40 Non Allele-specific 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 mouse IL-12/IL-23 p40 (non-allele specific). 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: IL-12/IL-23 p40
Interleukin 12 (IL-12), also known as natural killer cell stimulatory factor (NKSF) or cytotoxic lymphocyte maturation factor (CLMF), is a heterodimeric pleiotropic cytokine made up of a 40 kDa (p40) subunit and a 35 kDa (p35) subunit. The IL-12 p40 subunit is shared by IL-23, another heterodimeric cytokine that has biological activities similar to, as well as distinct from, IL-12. IL-12 is produced by macrophages and B cells and has been shown to have multiple effects on T cells and natural killer (NK) cells. While mouse IL-12 is active on both human and mouse cells, human IL-12 is not active on mouse 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 Mouse IL-12/IL-23 p40 Non Allele-specific 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 - 10
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Sphinganine recruits TLR4 adaptors in macrophages and promotes inflammation in murine models of sepsis and melanoma
Authors: Hering, M;Madi, A;Sandhoff, R;Ma, S;Wu, J;Mieg, A;Richter, K;Mohr, K;Knabe, N;Stichling, D;Poschet, G;Bestvater, F;Frank, L;Utikal, J;Umansky, V;Cui, G;
Nature communications
Species: Mouse
Sample Types: Cell Culture Supernates, Plasma
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Skeletal muscle antagonizes antiviral CD8+ T cell exhaustion
Authors: J Wu, N Weisshaar, A Hotz-Wagen, A Madi, S Ma, A Mieg, M Hering, K Mohr, T Schlimbach, H Borgers, G Cui
Sci Adv, 2020-06-12;6(24):eaba3458.
Species: Mouse
Sample Types: Serum
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Cysteine-Reactive Free ISG15 Generates IL-1?-Producing CD8?+ Dendritic Cells at the Site of Infection
Authors: A Napolitano, AG van der Ve, M Bunyan, A Borg, D Frith, S Howell, S Kjaer, A Beling, AP Snijders, KP Knobeloch, EM Frickel
J. Immunol., 2018-06-11;0(0):.
Species: Mouse
Sample Types: Serum
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STAT1 Represses Cytokine-Producing Group 2 and Group 3 Innate Lymphoid Cells during Viral Infection
Authors: MT Stier, K Goleniewsk, JY Cephus, DC Newcomb, TP Sherrill, KL Boyd, MH Bloodworth, ML Moore, K Chen, JK Kolls, RS Peebles
J. Immunol., 2017-06-02;0(0):.
Species: Mouse
Sample Types: Tissue Homogenates
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Tumor Microenvironment Remodeling by 4-Methylumbelliferone Boosts the Antitumor Effect of Combined Immunotherapy in Murine Colorectal Carcinoma.
Authors: Malvicini M, Fiore E, Ghiaccio V, Piccioni F, Rizzo M, Olmedo Bonadeo L, Garcia M, Rodriguez M, Bayo J, Peixoto E, Atorrasagasti C, Alaniz L, Aquino J, Matar P, Mazzolini G
Mol Ther, 2015-06-24;23(9):1444-55.
Species: Mouse
Sample Types: Tissue Homogenates
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Transcriptional analysis of murine macrophages infected with different Toxoplasma strains identifies novel regulation of host signaling pathways.
Authors: Melo M, Nguyen Q, Cordeiro C, Hassan M, Yang N, McKell R, Rosowski E, Julien L, Butty V, Darde M, Ajzenberg D, Fitzgerald K, Young L, Saeij J
PLoS Pathog, 2013-12-19;9(12):e1003779.
Species: Mouse
Sample Types: Cell Culture Supernates
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Continuous and discontinuous cigarette smoke exposure differentially affects protective Th1 immunity against pulmonary tuberculosis.
Authors: Shaler C, Horvath C, McCormick S, Jeyanathan M, Khera A, Zganiacz A, Kasinska J, Stampfli M, Xing Z
PLoS ONE, 2013-03-19;8(3):e59185.
Species: Mouse
Sample Types: Cell Culture Supernates
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Central role of MyD88-dependent dendritic cell maturation and proinflammatory cytokine production to control Brucella abortus infection.
Authors: Macedo GC, Magnani DM, Carvalho NB, Bruna-Romero O, Gazzinelli RT, Oliveira SC
J. Immunol., 2008-01-15;180(2):1080-7.
Species: Mouse
Sample Types: Cell Culture Supernates
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Mycobacterium tuberculosis antigens specifically modulate CCR2 and MCP-1/CCL2 on lymphoid cells from human pulmonary hilar lymph nodes.
Authors: Arias MA, Jaramillo G, Lopez YP, Mejia N, Mejia C, Pantoja AE, Shattock RJ, Garcia LF, Griffin GE
J. Immunol., 2007-12-15;179(12):8381-91.
Species: Human
Sample Types: Cell Culture Supernates
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IL-27 synthesis induced by TLR ligation critically depends on IFN regulatory factor 3.
Authors: Molle C, Nguyen M, Flamand V, Renneson J, Trottein F, De Wit D, Willems F, Goldman M, Goriely S
J. Immunol., 2007-06-15;178(12):7607-15.
Species: Mouse
Sample Types: Cell Culture Supernates
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Nod1-mediated innate immune recognition of peptidoglycan contributes to the onset of adaptive immunity.
Authors: Fritz JH, Le Bourhis L, Sellge G, Magalhaes JG, Fsihi H, Kufer TA, Collins C, Viala J, Ferrero RL, Girardin SE, Philpott DJ
Immunity, 2007-04-12;26(4):445-59.
Species: Mouse
Sample Types: Cell Culture Supernates
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Interleukin 10 regulates inflammatory cytokine synthesis to protect against lipopolysaccharide-induced abortion and fetal growth restriction in mice.
Authors: Robertson SA, Care AS, Skinner RJ
Biol. Reprod., 2007-01-10;76(5):738-48.
Species: Mouse
Sample Types: Serum
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Essential role for IL-10 in resistance to lipopolysaccharide-induced preterm labor in mice.
Authors: Robertson SA, Skinner RJ, Care AS
J. Immunol., 2006-10-01;177(7):4888-96.
Species: Mouse
Sample Types: Serum
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