Human IL-1ra/IL-1F3 DuoSet ELISA

Catalog # Availability Size / Price Qty
DY280
DY280-05
Ancillary Products Available
Human IL-1ra / IL-1F3 ELISA Standard Curve
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Product Details
Procedure
Citations (15)
FAQs
Supplemental Products
Reviews (3)

Human IL-1ra/IL-1F3 DuoSet ELISA Summary

Assay Type
Solid Phase Sandwich ELISA
Format
96-well strip plate
Sample Volume Required
100 µL
Sufficient Materials
For five or 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 human IL-1ra/IL-1F3. 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)

Data Example

Human IL-1ra / IL-1F3 ELISA Standard Curve

Product Datasheets

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-1ra/IL-1F3

Interleukin-1 receptor antagonist (IL-1ra; also known as IL-1F3) is a 22-25 kDa member of the IL-1 family of cytokines. Currently, there are 11 family members (IL-1F1-F11), nine of which form an IL-1 gene cluster on human Ch2 (1-3). Each IL-1 family member contains an IL-1 fold. This fold is generated by 12 packed beta -sheets that interact to form a beta -trefoil structure. Little amino acid (aa) homology is required to achieve this structure, and this explains the low aa identity among IL-1 family members. IL-1ra is a pure cytokine receptor antagonist that has no signal transduction-initiating activity (4). It is an acute phase protein that exists to dampen inflammation. IL-1( beta ) is initially produced by monocytes in response to a variety of stimuli. Circulating IL-1 then binds to widely expressed IL-1 type I receptors (IL-1 RI) and initiates a number of pro-inflammatory events. On endothelial cells (EC), IL-1 induces PGE2 and IL-6 release, generating fever, thrombocytosis, and hepatic acute phase protein production. In synovial joints, IL-1 induces chondrocyte NO production, an event that leads to reduced collagen synthesis and chondrocyte apoptosis. Finally, IL-1 increases neutrophil counts, both in blood and tissue, and thus is able to promote a pro-inflammatory environment in multiple locations (5-8). IL-1ra blocks IL-1 action through competitive inhibition. More correctly, although IL-1ra fills the IL-1 binding site in IL-1 RI, it is also unable to orchestrate the creation of a signal-transducing IL-1 RI:IL-1 R Accessory protein (IL-1 R AcP) heterodimer complex. Effective IL-1ra concentrations are generally 100-fold greater than local IL-1 concentrations. This is because the IL-1ra half-life is but 6 minutes, and very few IL-1 type I receptors need to be engaged by IL-1 to elicit a cellular response (5, 7, 9). 

Human IL-1ra is synthesized as a 177 aa precursor that contains a 25 aa signal sequence and a 152 aa mature region (10, 11). Although it contains an IL-1 cytokine fold, it apparently lacks two structural motifs that allow for activation of the IL-1 receptor heterodimer. First, and following binding to IL-1 RI, the presence of Ile 51-His 54 and Lys 145 of the mature molecule preclude recruitment of IL-1 R AcP. Second, there is no identifiable C-terminal lectin segment that is hypothesized to help recruit an accessory signaling component (1, 12, 13). Mature human IL-1ra is 77% and 82% aa identical to mouse and canine IL-1ra, respectively, and human IL-1ra inhibits IL-1 activity on mouse cells (10). A number of cell types express IL-1ra, including monocytes (11), Sertoli cells (14), hepatocytes (15), adipocytes (16), synovial fibroblasts (17), mast cells (18), pancreatic beta -cells (19), and intestinal epithelial cells (20). There are at least three intracellular IL-1ra isoforms (icIL-1ra1, 2, and 3). All show N-terminal variation, and all contain amino acids 35-177 of the secreted precursor (21-23). Intracellular IL-1ra1 is of particular interest, because it is reported to be "secreted" by endothelial cells and binds to the IL-1 RI in an antagonist fashion (23-25). Intracellular IL-1ra1 is 159 aa in length and shows a 3 aa substitution for the first 21 aa's of the signal sequence of IL-1ra (21).

Long Name:
Interleukin 1 Receptor Antagonist
Entrez Gene IDs:
3557 (Human); 16181 (Mouse); 60582 (Rat); 397499 (Porcine)
Alternate Names:
DIRA; ICIL-1ra; IL1F3; IL-1F3; IL1ra; IL-1ra; IL-1ra3; IL1RN; IL-1RN; interleukin 1 receptor antagonist; IRAP; MVCD4

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 Human IL-1ra/IL-1F3 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.

15 Citations: Showing 1 - 10
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  1. IL-1RA is part of the inflammasome-regulated immune response in bladder epithelial cells and influences colonization of uropathogenic E. coli
    Authors: A Lindblad, K Persson, I Demirel
    Cytokine, 2019;123(0):154772.
    Species: Human
    Sample Types: Cell Culture Supernates
  2. Local oestrogen therapy modulates extracellular matrix and immune response in the vaginal tissue of post-menopausal women with severe pelvic organ prolapse
    Authors: T Tyagi, M Alarab, Y Leong, S Lye, O Shynlova
    J. Cell. Mol. Med., 2019;0(0):.
    Species: Human
    Sample Types: Tissue Homogenates
  3. PASylation of IL-1 Receptor antagonist (IL-1Ra) retains IL-1 blockade and extends its duration in mouse urate crystal-induced peritonitis
    Authors: NE Powers, B Swartzwelt, C Marchetti, DM de Graaf, A Lerchner, M Schlapschy, R Datar, U Binder, CK Edwards, A Skerra, CA Dinarello
    J. Biol. Chem., 2019;0(0):.
    Species: Human
    Sample Types: Plasma
  4. The Phenotype and Secretory Activity of Adipose-Derived Mesenchymal Stem Cells (ASCs) of Patients with Rheumatic Diseases
    Authors: E Kuca-Warna, U Skalska, I Janicka, U Musia?owic, K Bonek, P G?uszko, P Szcz?sny, M Olesi?ska, E Kontny
    Cells, 2019;8(12):.
    Species: Human
    Sample Types: Cell Culture Superantes
  5. Production and regulation of interleukin-1 family cytokines at the materno-fetal interface
    Authors: LM Scott, AH Bryant, A Rees, B Down, RH Jones, CA Thornton
    Cytokine, 2017;0(0):.
    Species: Human
    Sample Types: Tissue Culture Supernates
  6. Interleukin-36 potently stimulates human M2 macrophages, Langerhans cells and keratinocytes to produce pro-inflammatory cytokines
    Authors: Damien Dietrich
    Cytokine, 2016;84(0):88-98.
    Species: Human
    Sample Types: Cell Culture Supernates
  7. Macrophage activation in acute exacerbation of idiopathic pulmonary fibrosis.
    Authors: Schupp J, Binder H, Jager B, Cillis G, Zissel G, Muller-Quernheim J, Prasse A
    PLoS ONE, 2015;10(1):e0116775.
    Species: Human
    Sample Types: BALF
  8. Phagocytosis of Staphylococcus aureus by human neutrophils prevents macrophage efferocytosis and induces programmed necrosis.
    Authors: Greenlee-Wacker M, Rigby K, Kobayashi S, Porter A, DeLeo F, Nauseef W
    J Immunol, 2014;192(10):4709-17.
    Species: Human
    Sample Types: Cell Culture Supernates
  9. Angiopoietin-1 upregulates de novo expression of IL-1beta and Il1-Ra, and the exclusive release of Il1-Ra from human neutrophils.
    Authors: Haddad L, Sirois M
    PLoS ONE, 2014;9(2):e88980.
    Species: Human
    Sample Types: Cell Lysates
  10. Effects of human Toll-like receptor 1 polymorphisms on ageing.
    Authors: Uciechowski P, Oellig E, Mariani E, Malavolta M, Mocchegiani E, Rink L
    Immun Ageing, 2013;10(1):4.
    Species: Human
    Sample Types: Plasma
  11. Regulation of progranulin expression in human microglia and proteolysis of progranulin by matrix metalloproteinase-12 (MMP-12).
    Authors: Suh HS, Choi N, Tarassishin L, Lee SC
    PLoS ONE, 2012;7(4):e35115.
    Species: Human
    Sample Types: Cell Culture Supernates
  12. Reprogramming of a subpopulation of human blood neutrophils by prolonged exposure to cytokines.
    Authors: Chakravarti A, Rusu D, Flamand N, Borgeat P, Poubelle PE
    Lab. Invest., 2009;89(10):1084-99.
    Species: Human
    Sample Types: Cell Culture Supernates
  13. The response of human epithelial cells to TNF involves an inducible autocrine cascade.
    Authors: Janes KA, Gaudet S, Albeck JG, Nielsen UB, Lauffenburger DA, Sorger PK
    Cell, 2006;124(6):1225-39.
    Species: Human
    Sample Types: Cell Culture Supernates
  14. Pre-interleukin-1alpha expression reduces cell growth and increases interleukin-6 production in SaOS-2 osteosarcoma cells: Differential inhibitory effect of interleukin-1 receptor antagonist (icIL-1Ra1).
    Authors: Palmer G, Trolliet S, Talabot-Ayer D, Mezin F, Magne D, Gabay C
    Cytokine, 2005;31(2):153-60.
    Species: Human
    Sample Types: Cell Culture Supernates
  15. Mice transgenic for intracellular interleukin-1 receptor antagonist type 1 are protected from collagen-induced arthritis.
    Authors: Palmer G, Talabot-Ayer D, Szalay-Quinodoz I, Maret M, Arend WP, Gabay C
    Eur. J. Immunol., 2003;33(2):434-40.
    Species: Human
    Sample Types: Serum

FAQs

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Reviews for Human IL-1ra/IL-1F3 DuoSet ELISA

Average Rating: 4.3 (Based on 3 Reviews)

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Human IL-1ra/IL-1F3 DuoSet ELISA
By Anonymous on 04/01/2018
Application: Sample Tested: Serum

Human IL-1ra/IL-1F3 DuoSet ELISA
By Anonymous on 03/19/2018
Application: Sample Tested: EDTA Plasma

Human IL-1ra/IL-1F3 DuoSet ELISA
By Anonymous on 08/29/2017
Application: Sample Tested: Serum

Very good ELISA.
We used human serum with no dilution. In our conditions, IL-1ra was not detectable in human seum.