Canine IL-6 DuoSet ELISA

Catalog # Availability Size / Price Qty
DY1609
Ancillary Products Available
Canine IL-6 ELISA Standard Curve
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Product Details
Procedure
Citations (9)
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Canine IL-6 DuoSet ELISA Summary

Assay Type
Solid Phase Sandwich ELISA
Format
96-well strip plate
Sample Volume Required
100 µL
Assay Range
62.5 - 4,000 pg/mL
Sufficient Materials
For fifteen 96-well plates*
Specificity
Please see the product datasheet

* 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 canine IL-6. 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

Canine IL-6 ELISA Standard Curve

Product Datasheets

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Preparation and Storage

Shipping
The product is shipped at ambient temperature. Upon receipt, store it immediately at the temperature recommended below.
Stability & Storage
Store the unopened product at 2 - 8 °C. Do not use past expiration date.

Background: IL-6

Interleukin 6 (IL-6) is a pleiotropic, alpha -helical, 22-28 kDa phosphorylated and variably glycosylated cytokine that plays important roles in the acute phase reaction, inflammation, hematopoiesis, bone metabolism, and cancer progression (1-5). Mature canine IL-6 is 187 amino acids (aa) in length and shares 58%, 37%, 39%, and 76% aa sequence identity with human, mouse, rat, and feline IL-6, respectively. Cells known to express IL-6 include CD8+ T cells, fibroblasts, synoviocytes, adipocytes, osteoblasts, megakaryocytes, endothelial cells (under the influence of endothelins), sympathetic neurons, cerebral cortex neurons, adrenal medulla chromaffin cells, retinal pigment cells, mast cells, keratinocytes, Langerhans cells, fetal and adult astrocytes, neutrophils, monocytes, eosinophils, colonic epithelial cells, B1 B cells and pancreatic islet beta cells (2, 6-28). IL-6 production is generally correlated with cell activation and is normally kept in control by glucocorticoids, catecholamines, and secondary sex steroids (2). Normal human circulating IL-6 is in the 1 pg/mL range, with slight elevations during the menstrual cycle, modest elevations in certain cancers, and large elevations after surgery (29-33). 

IL-6 induces signaling through a cell surface heterodimeric receptor complex composed of a ligand binding subunit (IL-6 R alpha) and a signal transducing subunit (gp130). IL-6 binds to IL-6 R alpha, triggering IL-6 R alpha association with gp130 and gp130 dimerization (34). gp130 is also a component of the receptors for CLC, CNTF, CT-1, IL-11, IL-27, LIF, and OSM (35). Soluble forms of IL-6 R alpha are generated by both alternative splicing and proteolytic cleavage (5). In a mechanism known as trans-signaling, complexes of soluble IL-6 and IL-6 R alpha elicit responses from gp130- expressing cells that lack cell surface IL-6 R alpha (5). Trans-signaling enables a wider range of cell types to respond to IL-6, as the expression of gp130 is ubiquitous, while that of IL-6 R alpha is predominantly restricted to hepatocytes, monocytes, and resting lymphocytes (2, 5). Soluble splice forms of gp130 block trans-signaling from IL-6/IL-6 R alpha but not from other cytokines that use gp130 as a co-receptor (5, 36). 
IL-6, along with TNF-alpha and IL-1, drives the acute inflammatory response. IL-6 is almost solely responsible for fever and the acute phase response in the liver, and it is important in the transition from acute inflammation to either acquired immunity or chronic inflammatory disease (1-5). When dysregulated, it contributes to chronic inflammation in conditions such as obesity, insulin resistance, inflammatory bowel disease, arthritis, and sepsis (2, 5). IL-6 modulates bone resorption and is a major effector of inflammatory joint destruction in rheumatoid arthritis through its promotion of Th17 cell development and activity (1). It contributes to atherosclerotic plaque development and destabilization as well as the development of inflammation-associated carcinogenesis (1, 2). IL-6 can also function as an anti-inflammatory molecule, as in skeletal muscle where it is secreted in response to exercise (2). In addition, it enhances hematopoietic stem cell proliferation and the differentiation of memory B cells and plasma cells (37). 

Long Name:
Interleukin 6
Entrez Gene IDs:
3569 (Human); 16193 (Mouse); 24498 (Rat); 399500 (Porcine); 280826 (Bovine); 403985 (Canine); 102138971 (Cynomolgus Monkey); 100034196 (Equine); 493687 (Feline); 463288 (Primate); 100008733 (Rabbit)
Alternate Names:
B-cell differentiation factor; B-cell stimulatory factor 2; BSF2; BSF-2; CDF; CTL differentiation factor ; HSF; hybridoma growth factor; IFNB2; IFN-beta-2; IL6; IL-6; Interferon beta-2; interleukin 6 (interferon, beta 2); interleukin BSF-2; interleukin-6; MGI-2A

Assay Procedure

GENERAL ELISA PROTOCOL

Plate Preparation

  1. 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.
  2. 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.
  3. Block plates by adding 300 μL Reagent Diluent to each well. Incubate at room temperature for a minimum of 1 hour.
  4. Repeat the aspiration/wash as in step 2. The plates are now ready for sample addition.

Assay Procedure

  1. 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.
  2. Repeat the aspiration/wash as in step 2 of Plate Preparation.
  3. 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.
  4. Repeat the aspiration/wash as in step 2 of Plate Preparation.
  5. 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.
  6. Repeat the aspiration/wash as in step 2.
  7. Add 100 μL of Substrate Solution to each well. Incubate for 20 minutes at room temperature. Avoid placing the plate in direct light.
  8. Add 50 μL of Stop Solution to each well. Gently tap the plate to ensure thorough mixing.
  9. 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 Canine IL-6 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.

9 Citations: Showing 1 - 9
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  1. miR-148a regulation interferes in inflammatory cytokine and parasitic load in canine leishmaniasis
    Authors: GT Rebech, JP Bragato, SF Costa, JH de Freitas, MO Dos Santos, MF Soares, FR Eugênio, PSP Dos Santos, VMF de Lima
    PloS Neglected Tropical Diseases, 2023-01-31;17(1):e0011039.
    Species: Canine
    Sample Types: Cell Culture Supernates
  2. Immortalised canine buccal epithelial cells' CXCL8 secretion is affected by allergen extracts, Toll-like receptor ligands, IL-17A and calcitriol
    Authors: M Pelst, C Höbart, H de Rooster, B Devriendt, E Cox
    Veterinary research, 2022-09-13;53(1):72.
    Species: Canine
    Sample Types: Cell Culture Supernates
  3. Effects of irradiation and leukoreduction on down regulation of CXCL-8 and storage lesion in stored canine whole blood
    Authors: H Yang, W Kim, J Bae, H Kim, S Kim, J Choi, J Park, DI Jung, H Koh, D Yu
    J. Vet. Sci., 2019-01-31;0(0):.
    Species: Canine
    Sample Types: Plasma
  4. Early activation of deleterious molecular pathways in the kidney in experimental heart failure with atrial remodeling
    Authors: T Ichiki, BK Huntley, GJ Harty, SJ Sangaralin, JC Burnett
    Physiol Rep, 2017-05-15;5(9):.
    Species: Canine
    Sample Types: Tissue Homogenates
  5. Targeted Doxorubicin Delivery to Brain Tumors via Minicells: Proof of Principle Using Dogs with Spontaneously Occurring Tumors as a Model
    Authors: JA MacDiarmid, V Langova, D Bailey, ST Pattison, SL Pattison, N Christense, LR Armstrong, VN Brahmbhatt, K Smolarczyk, MT Harrison, M Costa, NB Mugridge, I Sedliarou, NA Grimes, DL Kiss, B Stillman, CL Hann, GL Gallia, RM Graham, H Brahmbhatt
    PLoS ONE, 2016-04-06;11(4):e0151832.
    Species: Canine
    Sample Types: Serum
  6. The lipopolysaccharide from Capnocytophaga canimorsus reveals an unexpected role of the core-oligosaccharide in MD-2 binding.
    Authors: Ittig S, Lindner B, Stenta M, Manfredi P, Zdorovenko E, Knirel YA, dal Peraro M, Cornelis GR, Zahringer U
    PLoS Pathog., 2012-05-03;8(5):e1002667.
    Species: Canine
    Sample Types: Cell Culture Supernates
  7. Assessment of urine solute and matrix effects on the performance of an enzyme-linked immunosorbent assay for measurement of interleukin-6 in dog urine.
    Authors: Wood MW, Nordone SK, Vaden SL, Breitschwerdt EB
    J. Vet. Diagn. Invest., 2011-03-01;23(2):316-20.
    Species: Canine
    Sample Types: Urine
  8. Autocrine effects of interleukin-6 mediate acute-phase proinflammatory and tissue-reparative transcriptional responses of canine bladder mucosa.
    Authors: Wood MW, Breitschwerdt EB, Gookin JL
    Infect. Immun., 2010-11-29;79(2):708-15.
    Species: Canine
    Sample Types: Cell Culture Supernates
  9. A functional comparison of canine and murine bone marrow derived cultured mast cells.
    Authors: Lin TY, London CA
    Vet. Immunol. Immunopathol., 2006-10-06;114(3):320-34.
    Species: Canine
    Sample Types: Cell Culture Supernates

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