Human EBI3 DuoSet ELISA

R&D Systems | Catalog # DY6456-05

R&D Systems
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Key Product Details

Assay Type

Solid Phase Sandwich ELISA

Assay Range

62.5-4000 pg/mL

Sample Type

Cell culture supernates, serum, and plasma
Note: Diluents for complex matrices, such as serum and plasma, should be evaluated prior to use in this DuoSet

Reactivity

Human

Human EBI3 DuoSet ELISA 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
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Product Summary for Human EBI3 DuoSet ELISA

This DuoSet ELISA Development kit contains the basic components required for the development of sandwich ELISAs to measure natural and recombinant human Epstein-Barr virus induced gene 3
(IL-27/IL-35 EBI3 Subunit). The suggested diluent is suitable for the analysis of most cell culture supernate, serum, and plasma samples. Diluents for complex matrices, such as serum and plasma, should be evaluated prior to use in this DuoSet.

Product Specifications

Assay Format

96-well strip plate (sold separately)

Detection Method

Colorimetric ELISA - 450nm (TMB)

Conjugate

Biotin

Label

HRP

Kit Contents for Human EBI3 DuoSet ELISA

  • Capture Antibody
  • Detection Antibody
  • Recombinant Standard
  • Streptavidin conjugated to horseradish-peroxidase (Streptavidin-HRP)

Other Reagents Required

DuoSet Ancillary Reagent Kit 2 (5 plates): (Catalog # DY008C) containing 96 well microplates, plate sealers, substrate solution, stop solution, plate coating buffer (PBS), wash buffer, and Reagent Diluent Concentrate 2.

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 equivalent

Reagent Diluent*

Blocking Buffer*

Substrate Solution: ELISA TMB Substrate (Catalog # DY999B or DY999B-250)

Stop Solution: Methanesulfonic acid (Catalog # DY994B or DY994B-250)

Microplates: (Catalog # DY990), or equivalent

Plate Sealers: (Catalog # DY992), or equivalent

*For the recommended Reagent Diluent and Blocking Buffer for a specific DuoSet ELISA Development Kit, refer to the product datasheet.

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: EBI3

Interleukin-27 (IL-27) is a heterodimeric group 2 receptor ligand molecule that belongs to the IL-6/IL-12 family of long type I cytokines. It is composed of EBI3 (EBV-induced gene 3), a 34 kDa glycoprotein that is related to the p40 subunit of IL-12 and IL-23, and p28, the 28 kDa glycoprotein that is related to the p35 chain of IL-12). The human EBI3 gene encodes a 229 amino acid (aa) precursor that contains a 20 aa signal peptide and 209 aa mature protein. The mature region contains two potential N-linked glycosylation sites, two fibronectin type III domains, and two pairs of conserved cysteine residues with a WSxWS-like motif that places the IL-27 molecule in the hematopoietin receptor family. Although p40, the EBI3 counterpart in IL-12, is known to form homodimers, there is no evidence to date that EBI3 also homodimerizes.

Human EBI3 is 61% aa identical to mouse EBI3. The human p28 gene encodes a 243 aa precursor that contains a 28 aa signal sequence and 215 aa mature region. The mature region is characterized by the presence of four α-helices, placing it in the IL-6 family of helical cytokines. Human p28 is 74% aa identical to mouse p28. IL-27 is expressed by monocytes, endothelial cells and dendritic cells. IL-27 binds to and signals through a heterodimeric receptor complex composed of WSX-1 (TCCR) and gp130. Evidence suggests IL-27 interacts only with WSX-1. IL-27 has both anti- and pro-inflammatory properties. As an anti-inflammatory, IL-27 seems to induce a general negative feedback program that limits T and NK-T cell activity. At the onset of infection, IL-27 induces an IL-12 receptor on naïve CD4+ T cells, making them susceptible to subsequent IL-12 activity (and possible Th1 development).

Long Name

Epstein-Barr virus induced gene 3

Alternate Names

EBI-3, IL-27 beta, IL-27B, IL-35 EBI3, IL35 EBI3

Entrez Gene IDs

10148 (Human); 50498 (Mouse); 680609 (Rat); 102144621 (Cynomolgus Monkey)

Gene Symbol

EBI3

Additional EBI3 Products

Product Documents for Human EBI3 DuoSet ELISA

Certificate of Analysis

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Note: Certificate of Analysis not available for kit components.

Product Specific Notices for Human EBI3 DuoSet ELISA

For research use only

Related Research Areas

Citations for Human EBI3 DuoSet ELISA

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Protocols

View specific protocols for Human EBI3 DuoSet ELISA (DY6456-05):

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.

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