Recombinant Mouse IL-27 (p28)

Recombinant mouse IL-27 p28 (also known as IL-30) is a valuable tool for immunological research due to its diverse and well-characterized biological activities across multiple immune cell populations and disease models.

Immune Cell Activation and Differentiation

Recombinant IL-27 p28 serves as a potent activator of innate and adaptive immune responses. The protein triggers rapid clonal expansion of antigen-specific naive CD4+ T cells and promotes early Th1 differentiation through upregulation of interferon-gamma (IFN-γ) production. Additionally, it enhances the cytotoxic activity of CD8+ T cells, with in vitro studies demonstrating that IL-27 acts directly on naive CD8 cells to generate cytotoxic T lymphocytes with elevated granzyme B expression. The cytokine also co-stimulates IFN-γ production by natural killer cells and monocytes, making it useful for studying proinflammatory immune activation.

Regulatory and Anti-inflammatory Functions

Beyond its proinflammatory roles, recombinant IL-27 p28 exhibits important anti-inflammatory activities that are critical for understanding immune homeostasis. The protein induces IL-10 production by both naive and memory T cells and activates regulatory T cells (Treg), while simultaneously suppressing Th17 cytokine secretion. This dual functionality makes it particularly valuable for investigating immune tolerance mechanisms and the balance between inflammatory and regulatory responses.

Cancer Immunotherapy Applications

Recombinant IL-27 shows significant promise in cancer research contexts. The cytokine demonstrates antitumor activity through antiangiogenic mechanisms, activating production of antiangiogenic chemokines such as IP-10/CXCL10 and MIG/CXCL9. In cancer vaccine models, recombinant IL-27 at 10 ng/mouse provided significant improvement in prophylactic protection, with ≥50% of mice remaining tumor-free at 60 days post-engraftment. The protein also directly inhibits B-cell acute lymphoblastic leukemia cell spreading by blocking angiogenesis and cell proliferation while inducing apoptosis.

Host Defense and Infection Studies

IL-27 plays a critical role in host defense against intracellular pathogens. Studies using IL-27 receptor-deficient mice demonstrate increased susceptibility to infections such as Leishmania major and Listeria monocytogenes due to impaired IFN-γ production from CD4+ T cells. This makes recombinant IL-27 p28 essential for investigating immune responses to intracellular infections and understanding the molecular mechanisms of pathogen control.

Autoimmune Disease Research

The protein is instrumental in autoimmune disease models. IL-27 receptor-deficient mice show hypersusceptibility to experimental autoimmune encephalomyelitis with increased IL-17-producing Th cells. Additionally, IL-27 p28 deficiency exacerbates autoimmune diseases in Aire-deficient mice through disruption of Th2 bias in CD4+ single-positive thymocytes, making recombinant IL-27 valuable for studying mechanisms of immune tolerance and autoimmune pathogenesis.

Pain Threshold Regulation

An emerging application involves pain physiology research. Endogenous IL-27 constitutively controls thresholds for thermal and mechanical sensation, and neutralization of IL-27 p28 induces long-lasting mechanical hypersensitivity, suggesting utility in studying nociception and pain-related immune mechanisms.

Technical Advantages

Recombinant IL-27 p28 is a well-characterized 28 kDa secreted protein that shares 70% and 89% amino acid sequence identity with human and rat p28, respectively, facilitating cross-species comparative studies. The protein's expression can be regulated through multiple signaling pathways including MyD88-mediated and interferon regulatory factor 1-dependent mechanisms, allowing researchers to investigate transcriptional regulation and signal transduction in detail.

Yes, recombinant Mouse IL-27 (p28) can be used as a standard for quantification or calibration in ELISA assays, provided it is properly validated and matched to the assay system. Recombinant IL-27 p28 is commonly used as a standard in commercial ELISA kits designed to quantify mouse IL-27 p28 in biological samples.

Key considerations and supporting details:

• Assay Compatibility: ELISA kits for mouse IL-27 p28 typically use recombinant mouse IL-27 p28 as the standard to generate the calibration curve. The standard curve is constructed by plotting absorbance values against known concentrations of recombinant IL-27 p28, allowing quantification of unknown samples by interpolation.

• Validation: It is essential that the recombinant protein used as a standard is of high purity and its concentration is accurately determined. Commercial kits calibrate their assays using highly purified recombinant mouse IL-27 p28, and results obtained with natural samples show parallelism with the standard curve generated from the recombinant standard.

• Specificity: Most sandwich ELISA kits for mouse IL-27 p28 are validated to recognize both natural and recombinant forms of the protein, ensuring that quantification using recombinant standards is accurate for endogenous IL-27 p28 in samples.

• Preparation: The recombinant standard should be reconstituted and diluted according to the ELISA kit protocol. Use only the recommended diluent and follow the specified concentration range for calibration.

• Research Use: Recombinant standards are intended for research use only and not for diagnostic applications.

Best Practices:

• Confirm that your recombinant Mouse IL-27 (p28) matches the form and purity used in the ELISA kit you are employing.
• Prepare the standard curve using serial dilutions within the assay’s validated range (e.g., 7.8–500 pg/mL or 15.6–1000 pg/mL, depending on the kit).
• Always run standards in parallel with your samples for accurate quantification.

Limitations:

• If using a recombinant standard outside of a validated kit, ensure its biological activity and immunoreactivity are comparable to the natural protein.
• Some recombinant proteins may contain carrier proteins or preservatives that could affect assay performance; use carrier-free preparations if required by your protocol.

Summary Table: Use of Recombinant Mouse IL-27 (p28) as ELISA Standard

In conclusion, recombinant Mouse IL-27 (p28) is suitable as a standard for ELISA quantification, provided it is validated for your assay system and prepared according to protocol.

Recombinant Mouse IL-27 (p28) has been validated for several key applications in published research, primarily in bioassays, in vivo studies, and cell differentiation protocols.

Validated Applications:

• Bioassays (in vitro):

  • Used to stimulate and analyze immune cell responses, including naïve CD4+ and CD8+ T cells, B cells, NK cells, mast cells, and monocytes.
  • Induces phosphorylation of STAT1 and STAT3 in T cells, promoting differentiation and cytokine production (e.g., IFN-γ, IL-10).
  • Applied in studies of Tr1 cell differentiation, regulatory T cell (Treg) activation, and suppression of Th17 cytokine secretion.
  • Used as a standard in immunoassays and binding assays to study protein-protein interactions.

• In Vivo Studies:

  • Administered to mice to modulate disease models such as experimental autoimmune encephalomyelitis (EAE), allergic airway inflammation, malaria infection, and cancer.
  • Demonstrated to dampen EAE severity, inhibit tumor growth, and induce regression of neuroblastoma tumors.
  • Used to study the regulatory effects on hematopoietic stem/progenitor cell (HSPC) differentiation during infection.

• Stem/Immune Cell Maintenance or Differentiation:

  • Validated for protocols inducing Tr1 cell differentiation and maintenance of immune cell subsets.
  • Shown to work well in inducing IFN-γ and IL-10 expression in naïve mouse T cells.

• Immunohistochemistry:

  • Applied in tissue-based studies for immunological and oncology research.

Additional Context:

• Mechanistic Studies: IL-27 (p28) has been used to dissect signaling pathways involving IL-27Rα and gp130, as well as to study antagonistic effects of IL-30 (p28 alone) versus the IL-27 heterodimer.
• Disease Models: Research includes autoimmune inflammation, infectious disease, and oncology, with IL-27 (p28) administration affecting disease progression and immune regulation.
• Protein-Protein Interaction: Used in binding assays to characterize receptor interactions and downstream signaling.

Summary Table of Validated Applications

These applications are supported by multiple peer-reviewed studies and are widely used in immunology, infectious disease, and oncology research.

To reconstitute and prepare Recombinant Mouse IL-27 (p28) protein for cell culture experiments, follow these steps:

• Reconstitution:
  Add sterile phosphate-buffered saline (PBS) to the lyophilized protein to achieve a final concentration of 100 μg/mL. If the formulation contains BSA as a carrier, ensure your PBS contains at least 0.1% human or bovine serum albumin to stabilize the protein. If the product is carrier-free, reconstitute directly in PBS.

• Procedure:

  1. Centrifuge the vial briefly before opening to collect all lyophilized material at the bottom.
  2. Add the calculated volume of sterile PBS (with or without carrier protein, as appropriate) to the vial.
  3. Gently pipette the solution down the sides of the vial to dissolve the protein.
     • Do not vortex; gentle mixing preserves protein integrity.
  4. Allow the vial to sit at room temperature for several minutes to ensure complete dissolution.

• Aliquoting and Storage:

  • After reconstitution, aliquot the solution into working volumes to avoid repeated freeze-thaw cycles.
  • Store aliquots at –20°C to –80°C for long-term use; short-term storage (up to one week) at 4°C is acceptable.
  • Avoid multiple freeze-thaw cycles, as this can degrade the protein.

• Dilution for Cell Culture:

  • Before adding to cell cultures, dilute the reconstituted stock to the desired working concentration using cell culture medium or PBS with 0.1% BSA to maintain stability.

Key technical notes:

• Always use sterile technique to prevent contamination.
• If the product datasheet specifies a different buffer (e.g., sterile water or Tris buffer), follow those instructions.
• For functional assays, confirm the final buffer composition is compatible with your cell culture system.

Summary Table:

These guidelines ensure optimal solubility, stability, and biological activity of recombinant mouse IL-27 (p28) for cell culture applications.