Recombinant Human IL-10 (aa 19-178)

Recombinant Human IL-10 (aa 19-178) is a valuable tool for research applications due to its well-characterized immunomodulatory properties and versatile biological functions.

Immunoregulatory Functions

Recombinant Human IL-10 (aa 19-178) serves as a key anti-inflammatory mediator with pleiotropic effects on immune responses. The protein functions as a homodimer with a molecular weight of 17-21 kD and exerts multiple regulatory effects across innate and adaptive immunity.

The recombinant form directly modulates T cell responses by enhancing interferon-gamma production in CD8+ T cells and stimulating their cytotoxic activity. Simultaneously, it suppresses excessive pro-inflammatory responses by inhibiting the synthesis of pro-inflammatory cytokines and downregulating antigen presentation capacity in monocytes and macrophages through reduced expression of MHC class II and costimulatory molecules.

B Cell and Humoral Responses

IL-10 (aa 19-178) enhances B cell proliferation and survival while increasing antibody production and immunoglobulin secretion. This dual effect on humoral immunity makes it particularly useful for studies examining antibody-mediated responses and B cell biology.

Signal Transduction and Molecular Mechanisms

The recombinant protein operates through well-defined signaling pathways. The IL-10 dimer binds to IL-10 receptor alpha chains, recruiting IL-10 receptor beta chains and activating a cascade involving JAK1, TYK2, and STAT3. Additionally, IL-10 can block NF-κB activity and regulate JAK-STAT signaling pathways, providing multiple molecular entry points for mechanistic studies.

Therapeutic Research Applications

Recombinant Human IL-10 (aa 19-178) has demonstrated therapeutic potential in cancer immunotherapy research. When PEGylated formulations are used, the protein shows enhanced ability to induce tumor rejection and develop immunological memory against tumor cells. The recombinant protein has also been evaluated as an adjuvant to enhance vaccine efficacy in preclinical and clinical studies.

Research Quality and Specifications

The recombinant protein is typically produced with high purity (>97% by SDS-PAGE) and is available in bioactive, validated forms suitable for bioassays and immunological studies. Its amino acid sequence (aa 19-178) represents the mature human IL-10 form, which shares substantial sequence identity with IL-10 from other mammalian species, enabling comparative studies.

Yes, recombinant human IL-10 (including the aa 19-178 form) can be used as a standard for quantification or calibration in ELISA assays, provided it is suitable for your specific assay format and detection system.

Key Points:

• Standard Use: Recombinant human IL-10 is commonly used as a quantitative standard in ELISA assays to generate a standard curve for accurate measurement of IL-10 concentrations in samples. This is true for both full-length and truncated forms (such as aa 19-178), as long as the epitopes recognized by your capture and detection antibodies are present in the recombinant protein.

• Purity and Endotoxin: For reliable results, ensure the recombinant IL-10 is highly purified (≥95% purity by SDS-PAGE) and has low endotoxin levels (≤0.1 ng/µg is typical for research-grade standards).

• Carrier Protein: If the recombinant IL-10 is supplied with a carrier protein (e.g., BSA), this is generally recommended for ELISA standards to prevent adsorption losses. Carrier-free versions may require the addition of a carrier protein (e.g., BSA at 5–10 mg/mL) for optimal stability and performance.

• Assay Compatibility: Confirm that your ELISA kit or antibody pair is compatible with the recombinant IL-10 standard. Most commercial ELISA kits are validated for use with recombinant human IL-10 standards, but always check the kit’s instructions or datasheet.

• Calibration: When using recombinant IL-10 as a standard, prepare a dilution series (e.g., doubling dilutions from 2,000 to 15 pg/mL) to generate a standard curve. This curve is used to interpolate the concentration of IL-10 in your samples.

Recommendations:

• Use the recombinant IL-10 standard in the same matrix as your samples (e.g., assay diluent or buffer) to minimize matrix effects.
• Follow the manufacturer’s instructions for reconstitution and dilution.
• For best results, use fresh dilutions and avoid repeated freeze-thaw cycles.

In summary, recombinant human IL-10 (aa 19-178) is suitable as a standard for ELISA quantification, provided it is compatible with your assay system and handled according to best practices.

Recombinant Human IL-10 (aa 19-178) has been validated for several key applications in published research, primarily in the context of immunology, inflammation, and cell signaling studies.

Validated Applications in Published Research:

• Bioassays:
  Used to assess biological activity, such as the induction of cell proliferation (e.g., MC/9 mast cell proliferation assays) and signaling through the IL-10 receptor pathway, including JAK-STAT activation. These assays confirm the protein’s functional integrity and its ability to modulate immune cell responses.

• ELISA Standard:
  Employed as a reference standard in ELISA (enzyme-linked immunosorbent assay) for the quantification of IL-10 in biological samples, including serum and cell culture supernatants. This application is critical for measuring endogenous or experimental IL-10 levels in various disease models and clinical samples.

• Reference Standard in Biomarker Detection:
  Used as a standard in multiplex or biochip-based assays for parallel detection of inflammatory biomarkers, such as in the diagnosis of systemic inflammatory response syndrome (SIRS).

• Cell Culture Studies:
  Applied to cell culture systems to study its effects on immune cell differentiation, cytokine production, and modulation of T cell and B cell responses. For example, it has been used to investigate the immunomodulatory effects of cancer-associated fibroblasts on T cells.

• Preclinical and Clinical Research:
  Systemic administration of recombinant human IL-10 has been tested in clinical trials for autoimmune and inflammatory diseases, including Crohn’s disease, rheumatoid arthritis, psoriasis, and inflammatory bowel disease. In these settings, the protein is used to evaluate therapeutic efficacy and safety.

Representative Published Studies:

• Bioassay and ELISA Standard:

  • Differential signaling of cmvIL-10 through common variants of the IL-10 receptor 1 (Eur. J. Immunol., 2008): Used for bioassays in whole cells.
  • Hydrogel based protein biochip for parallel detection of biomarkers for diagnosis of SIRS in human serum (PLoS ONE, 2019): Used as an ELISA standard in serum samples.
  • Human Pancreatic Carcinoma-Associated Fibroblasts Promote Expression of Co-inhibitory Markers on CD4+ and CD8+ T-Cells (Frontiers in Immunology): Used as an ELISA standard in cell culture supernatants.

• Functional and Mechanistic Studies:

  • The vitamin D analogue calcipotriol promotes an anti-tumorigenic phenotype of human pancreatic CAFs but reduces T cell mediated immunity (Sci Rep, 2020): Used as a reference standard in functional studies.

Summary Table of Validated Applications

Additional Context:

• IL-10 (aa 19-178) is the mature, biologically active form of the cytokine, and its applications leverage its anti-inflammatory and immunoregulatory properties.
• It is widely used in both basic research and translational/clinical studies to dissect IL-10’s role in immune modulation, disease pathogenesis, and therapeutic intervention.

If you need protocols or more detailed application notes for a specific assay, please specify the intended use.

To reconstitute and prepare Recombinant Human IL-10 (aa 19-178) for cell culture experiments, dissolve the lyophilized protein at a concentration of 10 μg/mL in sterile PBS containing at least 0.1% human or bovine serum albumin (BSA or HSA). This ensures protein stability and prevents adsorption to surfaces.

Step-by-step protocol:

• Before opening the vial: Briefly centrifuge to collect all powder at the bottom.
• Reconstitution: Add sterile PBS (pH 7.2–7.4) with 0.1% albumin to achieve the desired concentration (typically 10 μg/mL for stock).
• Mixing: Gently swirl or tap the vial to dissolve. Do not vortex or mix vigorously, as this may denature the protein.
• Incubation: If solubility issues arise, allow the solution to incubate at 4°C overnight.
• Aliquoting: Divide into single-use aliquots to avoid repeated freeze-thaw cycles, which can degrade the protein.
• Storage: Store aliquots at −20°C to −70°C in a manual defrost freezer. After reconstitution, short-term storage (up to 1 month) at 2–8°C is acceptable under sterile conditions.
• Working solution: For cell culture, dilute the stock in culture medium (e.g., RPMI + 10% FBS) to the final working concentration (e.g., 20 ng/mL for THP-1 cell polarization).

Additional notes:

• Always use endotoxin-free reagents and sterile technique.
• Albumin (BSA or HSA) is critical to prevent loss of protein due to adsorption and to stabilize the cytokine in solution.
• Avoid reconstituting at concentrations above 1 mg/mL to prevent solubility issues.
• If the protein appears as a film, gently rinse the vial walls with buffer to recover all material.
• Confirm protein integrity by SDS-PAGE if needed.

This protocol is suitable for most cell culture applications involving recombinant IL-10 (aa 19-178). Adjust final working concentrations according to experimental requirements.