Recombinant Human EBI3 (IL-27 / IL-35)

Recombinant Human EBI3 (IL-27/IL-35) is used in research applications to study immune regulation, cytokine biology, and disease mechanisms, as well as to explore its therapeutic potential in inflammation, infection, autoimmunity, and cancer.

Key reasons to use recombinant EBI3 in your research include:

• Cytokine Subunit Function: EBI3 is a critical subunit of the heterodimeric cytokines IL-27 (EBI3 + p28) and IL-35 (EBI3 + p35), both of which have potent immunomodulatory and anti-inflammatory properties. IL-27 and IL-35 regulate T cell responses, suppress excessive inflammation, and play roles in immune tolerance and tumor immunity.

• Immunomodulatory Effects: Recombinant EBI3 allows you to dissect its role in modulating immune responses, including its effects on T cells, NK cells, and dendritic cells. For example, EBI3 can dampen immune responses to viral infections and regulate cytokine production, such as IL-10 and IL-17A.

• Chaperone-like Activity: EBI3 has a unique intracellular role as a chaperone, promoting the proper folding and secretion of its partner subunits (notably p28 for IL-27), which is essential for the biological activity of these cytokines. Studying recombinant EBI3 can help elucidate protein folding and secretion mechanisms in the endoplasmic reticulum.

• Standalone Biological Activity: Recent studies indicate that EBI3, even as a monomer, may have independent anti-inflammatory effects and can modulate signaling pathways, such as weakly inducing STAT1 signaling. This opens avenues for research into EBI3’s direct effects outside of its role in heterodimeric cytokines.

• Disease Relevance: EBI3 and its cytokine complexes are implicated in a wide range of diseases, including autoimmune disorders, infectious diseases (notably Epstein-Barr virus-related conditions), inflammatory diseases, and various cancers. Recombinant EBI3 is a valuable tool for modeling these diseases and testing therapeutic hypotheses.

• Therapeutic Potential: Ongoing research and clinical trials are investigating recombinant EBI3 and its cytokine complexes as potential immunotherapeutic agents for cancer, autoimmunity, and chronic infections, due to their ability to modulate immune responses and promote immune tolerance.

• Experimental Versatility: Recombinant EBI3 can be used in:

  • In vitro cell culture assays to study cytokine signaling and immune cell differentiation.
  • In vivo models to assess its effects on disease progression, immune modulation, and therapeutic efficacy.
  • Protein interaction and structural studies to understand cytokine assembly and receptor binding.

In summary, recombinant human EBI3 is a versatile and essential reagent for investigating cytokine biology, immune regulation, and disease mechanisms, and for developing novel immunotherapies targeting inflammation, infection, and cancer.

Recombinant Human EBI3 (IL-27/IL-35) protein can be used as a standard for quantification or calibration in ELISA assays, provided it is properly validated for your specific assay format and detection antibodies.

Key considerations and supporting details:

• Use as Standard: Recombinant EBI3 is commonly used as a standard in ELISA assays designed to quantify EBI3 in biological samples. Commercial ELISA kits for EBI3 typically include recombinant EBI3 as the calibration standard, and development kits (e.g., DuoSet) are specifically intended for quantitative measurement using recombinant EBI3.

• Assay Type: If your ELISA is designed to detect EBI3 alone (as a monomer or subunit), recombinant EBI3 is appropriate as a standard. If your assay is intended to quantify heterodimeric cytokines such as IL-27 or IL-35, which consist of EBI3 paired with another subunit (IL-27p28 or IL-12p35, respectively), you must ensure your antibodies specifically recognize the heterodimer and not just the EBI3 subunit. In such cases, a recombinant heterodimer (IL-27 or IL-35) is preferred as the standard.

• Protein Formulation: Carrier-free recombinant EBI3 is recommended for ELISA standards to avoid interference from stabilizers like BSA, unless your assay tolerates carrier proteins.

• Validation: Before using recombinant EBI3 as a standard, validate its performance in your assay by confirming:

  • The standard curve is linear and covers the expected concentration range.
  • The recombinant protein is recognized by your capture and detection antibodies.
  • The standard reflects the native protein’s behavior in your sample matrix.

• Application Example: Published protocols and commercial kits demonstrate successful quantification of EBI3 using recombinant EBI3 as a standard, with sensitivity down to picogram levels. For IL-35 quantification, a sandwich ELISA using antibodies against both subunits (EBI3 and IL-12p35) and recombinant IL-35 as standard is required for specificity.

Summary Table: EBI3 Standard Use in ELISA

Conclusion:
You can use recombinant human EBI3 as a standard for ELISA quantification of EBI3, but for assays targeting IL-27 or IL-35 heterodimers, use the appropriate recombinant heterodimer as the standard and validate antibody specificity accordingly.

Research Applications and Validation

Recombinant human EBI3 has been validated and applied across multiple research domains, reflecting its diverse biological roles:

Immunomodulatory Studies

EBI3 serves as a critical tool for investigating immune regulation mechanisms. As a subunit of the heterodimeric cytokines interleukin-27 (IL-27) and interleukin-35 (IL-35), recombinant EBI3 enables researchers to study anti-inflammatory and immunosuppressive pathways. The protein has been validated for functional assays examining its capacity to modulate immune responses, particularly in contexts involving viral infections and immune dysregulation.

Viral Infection Research

Recombinant EBI3 has been applied to investigate immune responses against Epstein-Barr virus (EBV) and other pathogens. Its role in anti-viral immunity makes it valuable for studying mechanisms underlying EBV-associated diseases, including infectious mononucleosis, nasopharyngeal carcinoma, and EBV-related lymphomas.

Cancer Biology Applications

EBI3 has been validated in cancer research contexts, particularly regarding tumor microenvironment interactions. Studies have demonstrated its involvement in cancer cell plasticity and metastatic colonization, with macrophage-secreted IL-35 (containing EBI3) shown to regulate cancer cell behavior in metastatic organs. Additionally, EBI3 expression has been implicated in lung cancer, colorectal cancer, and acute myeloid leukemia research.

Protein Folding and Chaperone Studies

Beyond its cytokine functions, recombinant EBI3 has been validated for investigating its intracellular chaperone-like role in promoting proper protein folding in the endoplasmic reticulum, working in collaboration with the lectin chaperone calnexin.

Immunological Assays

EBI3 protein has been validated as a control reagent in immunological assays, including use with polyclonal or monoclonal anti-EBI3 antibodies. It is suitable for bioactivity assays and serves as a standard for validating antibody specificity.

Regulatory T Cell and Immune Tolerance Research

Recombinant IL-35 (containing EBI3) has been validated for studying regulatory T cell (Treg) populations and the induction of IL-35-producing induced regulatory T cells (iTr35). This application extends to investigating immune tolerance mechanisms in both physiological and pathological conditions.

Reconstitution Guidelines

Recombinant Human EBI3 protein is supplied in lyophilized form and requires proper reconstitution before use in cell culture experiments. The reconstitution process is critical for maintaining protein integrity and biological activity.

Initial Preparation Steps

Before reconstituting, centrifuge the vial to ensure all lyophilized material is at the bottom. When opening the vial, handle it carefully to prevent loss of the dried protein. Upon reconstitution, gently pipette the reconstitution solution down the sides of the vial to ensure complete recovery of the protein into solution. Do not vortex the sample, as this can cause protein denaturation and aggregation.

Reconstitution Buffers and Concentrations

EBI3 can be reconstituted in several different buffer systems depending on your experimental requirements:

Recommended Buffer Options

• PBS (Phosphate-Buffered Saline): Reconstitute at 200 μg/mL in sterile PBS
• Acetic Acid: Reconstitute in sterile 10 mM acetic acid at a concentration not less than 100 μg/mL
• Hydrochloric Acid: Reconstitute at 0.1 mg/mL in 10 mM HCl
• Alternative Acidic Solutions: Sterile 20 mM HCl at 0.1 mg/mL can also be used

After initial reconstitution, the protein can be further diluted into other aqueous solutions as needed for your specific experimental applications.

Storage and Stability

Lyophilized Protein Storage

Store the unopened, lyophilized vial at −20°C for up to one year.

Post-Reconstitution Storage

Once reconstituted, store the protein at 2–8°C (refrigerated conditions) for up to one week. For longer-term storage beyond one week, prepare aliquots and freeze at −20°C. When storing reconstituted protein for extended periods, consider adding a carrier protein such as 0.1% human serum albumin (HSA) or bovine serum albumin (BSA) to enhance stability and prevent non-specific adsorption to container surfaces.

Critical Handling Precautions

Use a manual defrost freezer and avoid repeated freeze-thaw cycles, as these can compromise protein structure and activity. When preparing working solutions for cell culture, further dilutions should be made in appropriate buffers containing carrier proteins such as 0.2–1% BSA or HSA.

Preparation for Cell Culture Applications

For cell culture experiments, prepare stock solutions at the recommended concentration and then dilute to your desired working concentration in culture medium or buffered solutions containing carrier protein. This approach minimizes protein degradation and maintains consistent biological activity across your experimental replicates. Ensure all solutions are sterile and handle the reconstituted protein aseptically to prevent contamination during cell culture work.