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Feline IL-6 DuoSet ELISA

Catalog #: DY2305
7 Citations 1 Review Datasheet / COA / SDS
Catalog # Availability Size / Price Qty
DY2305
Ancillary Products Available
DY008B: DuoSet ELISA Ancillary Reagent Kit 2
DY999B: Substrate Reagent Pack
DY999: Substrate Reagent Pack
Feline IL-6 ELISA Standard Curve
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Product Details
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Citations (7)
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Supplemental Products
Reviews (1)
Summary Product Features Kit Content Other Reagents Required Data Examples Product Datasheets Preparation and Storage Background

Feline IL-6 DuoSet ELISA Summary

Assay Type
Solid Phase Sandwich ELISA
Format
96-well strip plate
Sample Volume Required
100 µL
Assay Range
31.2 - 2,000 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 feline 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

N/A Feline IL-6 ELISA Standard Curve View Larger

Feline 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). Alternative splicing generates several isoforms with internal deletions, some of which exhibit antagonistic properties (7-10). 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, 11-33). IL-6 production is generally correlated with cell activation and is normally kept in control by glucocorticoids, catecholamines, and secondary sex steroids (2). 

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 (39). gp130 is also a component of the receptors for CLC, CNTF, CT-1, IL-11, IL-27, LIF, and OSM (40). 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, 41).
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 (42).

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 Feline 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.

7 Citations: Showing 1 - 7
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  1. Clinical and pathological findings in fatal cases of severe fever with thrombocytopenia syndrome with high viremia in cats
    Authors: A Matsuu, H Hatai, T Hifumi, E Hamakubo, M Take, T Tanaka, Y Momoi, Y Endo, A Koyoshi, Y Kamikubo, R Kamatsuki, N Kurusu, H Tabata, H Matsuyama, M Yanaidani, M Kawabata, T Kawabata
    Topics in companion animal medicine, 2022-12-28;52(0):100756.
    Species: Feline
    Sample Types: Serum
  2. Clinical Relevance of the serum CTLA-4 in Cats with Mammary Carcinoma
    Authors: AC Urbano, C Nascimento, M Soares, J Correia, F Ferreira
    Sci Rep, 2020-03-02;10(1):3822.
    Species: Feline
    Sample Types: Serum
  3. Adipokines secretion in feline primary adipose tissue culture in response to dietary fatty acids
    Authors: M Mazaki-Tov, SR Bolin, PA Schenck
    BMC Vet. Res., 2019-09-06;15(1):324.
    Species: Feline
    Sample Types: Cell Culture Supernates
  4. Human and feline adipose-derived mesenchymal stem cells have comparable phenotype, immunomodulatory functions, and transcriptome
    Authors: KC Clark, FA Fierro, EM Ko, NJ Walker, B Arzi, CG Tepper, H Dahlenburg, A Cicchetto, A Kol, L Marsh, WJ Murphy, N Fazel, DL Borjesson
    Stem Cell Res Ther, 2017-03-20;8(1):69.
    Species: Feline
    Sample Types: Cell Culture Supernates
  5. Failure of a dietary model to affect markers of inflammation in domestic cats.
    Authors: Verbrugghe A, Janssens G, Van de Velde H, Cox E, De Smet S, Vlaeminck B, Hesta M
    BMC Vet Res, 2014-05-04;10(0):104.
    Species: Feline
    Sample Types: Serum
  6. Systemic response to low-dose endotoxin infusion in cats.
    Authors: Declue AE, Williams KJ, Sharp C, Haak C, Lechner E, Reinero CR
    Vet. Immunol. Immunopathol., 2009-06-11;0(0):.
    Species: Feline
    Sample Types: Plasma
  7. Activation of p38 MAPK by feline infectious peritonitis virus regulates pro-inflammatory cytokine production in primary blood-derived feline mononuclear cells.
    Authors: Regan AD, Cohen RD, Whittaker GR
    Virology, 2008-12-05;384(1):135-43.
    Species: Feline
    Sample Types: Cell Culture Supernates

FAQs

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Feline IL-6 DuoSet ELISA
By Ashley Franklin on 08/10/2016
Sample Tested: Cell culture supernatant,Serum

This product was able to detect biologically relevant IL-6 concentrations in cell culture supernatants after PBMC stimulation with LPS, however percent recovery in serum was very low (only 7% with 1:2 dilution of serum). There were also issues for parallelism between serial dilutions of pooled cheetah serum and the standard. This product is valid for use with cell culture supernatants but I would not recommend it for detection of IL-6 in serum samples.

Feline IL-6 DuoSet ELISA DY2305