Porcine IL-12/IL-23 p40 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 porcine IL-12/IL-23 p40. 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 Porcine IL-12/IL-23 p40 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 - 8
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Early Immune Initiation by Porcine Cells following Toxoplasma gondii Infection versus TLR Ligation
Authors: B Hamid, J Schlosser-, L Bechtold, F Ebner, S Rausch, S Hartmann
Microorganisms, 2021-08-28;9(9):.
Species: Porcine
Sample Types: Cell Culture Supernates
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The Effect of Zearalenone on the Cytokine Environment, Oxidoreductive Balance and Metabolism in Porcine Ileal Peyer's Patches
Authors: K Obremski, W Trybowski, P Wojtacha, M Gaj?cka, J Tyburski, ? Zielonka
Toxins (Basel), 2020-05-27;12(6):.
Species: Porcine
Sample Types: Whole Tissue
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Intra-Species and Inter-Species Differences in Cytokine Production by Porcine Antigen-Presenting Cells Stimulated by Mycoplasma hyopneumoniae, M. hyorhinis, and M. flocculare
Authors: S Fourour, C Marois-Cré, L Martelet, C Fablet, I Kempf, M Gottschalk, M Segura
Pathogens, 2019-03-16;8(1):.
Species: Porcine
Sample Types: Cell Culture Supernates
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Plant synthetic GP4 and GP5 proteins from porcine reproductive and respiratory syndrome virus elicit immune responses in pigs
Authors: CH An, S Nazki, SC Park, YJ Jeong, JH Lee, SJ Park, A Khatun, WI Kim, YI Park, JC Jeong, CY Kim
Planta, 2018-01-08;0(0):.
Species: Porcine
Sample Types: Serum
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Early Gut Microbiota Intervention Suppresses DSS-Induced Inflammatory Responses by Deactivating TLR/NLR Signalling in Pigs
Authors: Y Xiao, H Yan, H Diao, B Yu, J He, J Yu, P Zheng, X Mao, Y Luo, D Chen
Sci Rep, 2017-06-12;7(1):3224.
Species: Porcine
Sample Types: Tissue Culture Supernates
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The immune response against Chlamydia suis genital tract infection partially protects against re-infection.
Authors: De Clercq E, Devriendt B, Yin L, Chiers K, Cox E, Vanrompay D
Vet Res, 2014-09-25;45(0):95.
Species: Porcine
Sample Types: Cell Culture Supernates
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Cytokine protein expression levels in tracheobronchial lymph node homogenates of pigs infected with pseudorabies virus.
Authors: Miller LC, Zanella EL, Waters WR
Clin. Vaccine Immunol., 2010-03-10;17(5):728-34.
Species: Porcine
Sample Types: Tissue Homogenates
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Although pig allogeneic mesenchymal stem cells are not immunogenic in vitro, intracardiac injection elicits an immune response in vivo.
Authors: Poncelet AJ, Vercruysse J, Saliez A, Gianello P
Transplantation, 2007-03-27;83(6):783-90.
Species: Porcine
Sample Types: Cell Culture Supernates
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