Human CCL26/Eotaxin-3 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 CCL26/Eotaxin-3. 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)
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
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 CCL26/Eotaxin-3 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.
11
Citations: Showing 1 - 10
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Tollip interaction with STAT3: a novel mechanism to regulate human airway epithelial responses to type 2 cytokines
Authors: N Schaunaman, KG Dimasuay, M Kraft, HW Chu
Respiratory Research, 2022-02-16;23(1):31.
Species: Human
Sample Types: Cell Culture Supernates
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One-Hour Esophageal String Test: A Nonendoscopic Minimally Invasive Test That Accurately Detects Disease Activity in Eosinophilic Esophagitis
Authors: SJ Ackerman, AF Kagalwalla, I Hirano, N Gonsalves, PM Katcher, S Gupta, JB Wechsler, M Grozdanovi, Z Pan, JC Masterson, J Du, RJ Fantus, P Alumkal, JJ Lee, S Ochkur, F Ahmed, K Capocelli, H Melin-Alda, K Biette, A Dubner, K Amsden, K Keeley, M Sulkowski, A Zalewski, D Atkins, GT Furuta
Am. J. Gastroenterol., 2019-10-01;114(10):1614-1625.
Species: Human
Sample Types: Tissue Homogenates
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Farnesyltransferase Inhibition Exacerbates Eosinophilic Inflammation and Airway Hyperreactivity in Mice with Experimental Asthma: The Complex Roles of Ras GTPase and Farnesylpyrophosphate in Type 2 Allergic Inflammation
Authors: JM Bratt, KY Chang, M Rabowsky, LM Franzi, SP Ott, S Filosto, T Goldkorn, M Arif, JA Last, NJ Kenyon, AA Zeki
J. Immunol., 2018-04-27;0(0):.
Species: Human
Sample Types: Cell Culture Supernates
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IL-13 induces periostin and eotaxin expression in human primary alveolar epithelial cells: Comparison with paired airway epithelial cells
Authors: Y Ito, R Al Mubarak, N Roberts, K Correll, W Janssen, J Finigan, R Mishra, HW Chu
PLoS ONE, 2018-04-19;13(4):e0196256.
Species: Human
Sample Types: Cell Culture Supernates
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Procyanidin A2 Modulates IL-4-Induced CCL26 Production in Human Alveolar Epithelial Cells
Int J Mol Sci, 2016-11-12;17(11):.
Species: Human
Sample Types: Cell Culture Supernates
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Eotaxin-3 as a Biomarker of Activity in Established Eosinophilic Granulomatosis with Polyangiitis
Authors: Pavel I Novikov
J. Rheumatol., 2016-11-01;43(11):2082-2083.
Species: Human
Sample Types: Serum
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JAK-STAT6 Pathway Inhibitors Block Eotaxin-3 Secretion by Epithelial Cells and Fibroblasts from Esophageal Eosinophilia Patients: Promising Agents to Improve Inflammation and Prevent Fibrosis in EoE
Authors: Edaire Cheng
PLoS ONE, 2016-06-16;11(6):e0157376.
Species: Human
Sample Types: Cell Culture Supernates
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Altered esophageal histamine receptor expression in Eosinophilic Esophagitis (EoE): implications on disease pathogenesis.
Authors: Merves J, Chandramouleeswaran P, Benitez A, Muir A, Lee A, Lim D, Dods K, Mehta I, Ruchelli E, Nakagawa H, Spergel J, Wang M
PLoS ONE, 2015-02-27;10(2):e0114831.
Species: Human
Sample Types: Cell Culture Supernates
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Neurotrophic tyrosine kinase receptor 1 is a direct transcriptional and epigenetic target of IL-13 involved in allergic inflammation.
Authors: Rochman M, Kartashov A, Caldwell J, Collins M, Stucke E, Kc K, Sherrill J, Herren J, Barski A, Rothenberg M
Mucosal Immunol, 2014-11-12;8(4):785-98.
Species: Human
Sample Types: Cell Lysates
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IL-13 involvement in eosinophilic esophagitis: transcriptome analysis and reversibility with glucocorticoids.
Authors: Blanchard C, Mingler MK, Vicario M, Abonia JP, Wu YY, Lu TX, Collins MH, Putnam PE, Wells SI, Rothenberg ME
J. Allergy Clin. Immunol., 2007-12-01;120(6):1292-300.
Species: Human
Sample Types: Cell Culture Supernates
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Eotaxin-3 and a uniquely conserved gene-expression profile in eosinophilic esophagitis.
Authors: Assa'ad AH
J. Clin. Invest., 2006-02-01;116(2):536-47.
Species: Human
Sample Types: Tissue Homogenates
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Average Rating: 5 (Based on 2 Reviews)
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We used this kit for the quantification of CCL26 in human serum and plasma. Works very well and well-described protocol.
