Recombinant Human IL-12 (sf Insect Cells)

Recombinant Human IL-12 produced in sf insect cells is a valuable tool for research applications requiring high-purity, biologically active cytokine to study immune modulation, anti-tumor responses, and hematopoietic recovery. Its use is particularly advantageous for experiments focused on cellular immunity, cancer immunotherapy, and infectious disease models.

Key scientific reasons to use this reagent include:

• Potent Immunomodulatory Activity: IL-12 is a critical cytokine for inducing Th1 differentiation, stimulating IFN-γ production, and enhancing the cytotoxic activity of NK and T cells. This makes it essential for studies investigating cellular immune responses, especially those targeting intracellular pathogens or tumor cells.

• Anti-Tumor and Anti-Metastatic Effects: Recombinant IL-12 has demonstrated strong anti-tumor activity in preclinical models, inhibiting tumor growth and metastasis through activation of cytotoxic lymphocytes and modulation of the tumor microenvironment.

• Hematopoietic Recovery and Radioprotection: IL-12 promotes recovery of hematopoietic function and regulates immunity following radiotherapy, making it useful for research on radiation-induced myelosuppression and immune reconstitution.

• Bridging Innate and Adaptive Immunity: IL-12 enhances antigen presentation by macrophages and dendritic cells, facilitating the activation and proliferation of T cells and NK cells, which is crucial for vaccine development and infectious disease research.

• Insect Cell Expression Advantages: Production in sf insect cells (baculovirus system) allows for efficient expression of complex, glycosylated proteins with proper folding and biological activity, often yielding high purity and low endotoxin levels suitable for sensitive immunological assays.

• Versatility in Experimental Design: Recombinant IL-12 can be used in vitro to stimulate immune cells, in vivo for animal models of cancer or infection, or as an adjuvant in vaccine studies to enhance Th1 responses.

When selecting recombinant human IL-12 from sf insect cells, you benefit from a protein that closely mimics native cytokine activity, is suitable for mechanistic studies, and supports translational research in immunology, oncology, and hematology. Always confirm purity, activity, and endotoxin levels for your specific application.

Yes, recombinant human IL-12 produced in Sf insect cells can be used as a standard for quantification or calibration in ELISA assays, provided it is properly validated and matched to your assay system.

Supporting details:

• Parallelism and Calibration: ELISA kits designed for human IL-12 quantification commonly use recombinant IL-12 expressed in Sf insect cells as their standard. Studies show that standard curves generated with recombinant IL-12 are parallel to those obtained with natural human IL-12, indicating comparable immunoreactivity and enabling accurate quantification.

• Assay Validation: The Quantikine Human IL-12 ELISA, for example, is calibrated against highly purified Sf21-expressed recombinant human IL-12 p70. The dose-response curve of the NIBSC/WHO International Standard 95/544 parallels the curve generated by the recombinant standard, allowing conversion between units if needed.

• Best Practices:

  • Ensure the recombinant IL-12 standard is of high purity and properly formulated (often with carrier protein such as BSA to maintain stability and prevent adsorption losses).
  • Validate that your ELISA antibodies recognize both recombinant and native IL-12 equivalently, as most commercial kits are designed for this.
  • Prepare a dilution series for your standard curve within the dynamic range of your assay, typically from low pg/mL to ng/mL concentrations.
  • Store and handle the recombinant standard according to manufacturer and protocol recommendations to preserve activity and consistency.

• Limitations:

  • Use only for research purposes, not for diagnostic procedures.
  • Do not mix standards or reagents from different lots or sources unless specifically validated for cross-compatibility.
  • If using a custom ELISA, confirm that your antibodies do not show differential affinity for insect cell-derived versus mammalian cell-derived IL-12.

Summary: Recombinant human IL-12 (sf insect cells) is widely accepted and validated as a standard for ELISA quantification of IL-12, provided it is matched to your assay system and handled according to best practices.

Recombinant Human IL-12 produced in insect (sf) cells has been validated in published research for several key applications, primarily in immunological and cancer-related studies.

Key validated applications include:

• In vitro functional assays: Recombinant IL-12 produced in insect cells has been used to stimulate proliferation of immune cell lines (such as CTLL-2) and to induce interferon-gamma (IFN-γ) production by spleen cells or NK cell lines, confirming its biological activity and utility in immune cell activation studies.
• Immunotherapy research: IL-12 produced in insect cells has been incorporated into fusion proteins and evaluated for its ability to modulate tumor microenvironments, reverse T cell anergy, and reprogram tumor-associated macrophages, supporting its use in preclinical immunotherapy models.
• ELISA and immunoassays: Recombinant human IL-12 from insect cells is validated for use as a standard or control in ELISA, chemiluminescent immunoassays (CLIA), and lateral flow assays, enabling quantification and detection of IL-12 in biological samples.
• Vaccine adjuvant studies: IL-12 has been used as an adjuvant to enhance cellular immune responses in vaccine research, particularly in models where strong Th1-type immunity is desired.

Supporting details:

• In vitro, insect cell-derived IL-12 has been shown to induce IFN-γ secretion in NK-92MI cells and to stimulate proliferation of T and NK cells, confirming its functional equivalence to IL-12 from other sources.
• In immunotherapy research, baculovirus-expressed IL-12 has been used to generate fusion proteins with tumor-targeting properties, demonstrating increased activity upon protease cleavage and supporting its use in targeted cytokine delivery strategies.
• Commercial and research-grade recombinant IL-12 from insect cells is routinely validated for immunoassays such as ELISA and CLIA, as well as for use in lateral flow detection platforms.

Summary of main validated applications:

If you need protocols or more specific details for any of these applications, please specify the intended use.

To reconstitute and prepare Recombinant Human IL-12 (sf Insect Cells) for cell culture experiments, dissolve the lyophilized protein in sterile phosphate buffered saline (PBS), pH 7.2–7.4, at a concentration of 0.2 mg/mL.

Essential steps and best practices:

• Centrifuge the vial briefly before opening to ensure all lyophilized material is at the bottom.
• Add the calculated volume of sterile PBS directly to the vial. For example, to achieve 0.2 mg/mL, add 500 μL PBS to a 0.1 mg vial.
• Gently mix by pipetting up and down or swirling; do not vortex, as vigorous mixing may denature the protein.
• If the protein appears as a thin film, ensure complete dissolution by gentle mixing and allow it to sit at room temperature for a few minutes.
• For extended storage, aliquot the reconstituted protein into smaller volumes to avoid repeated freeze-thaw cycles.
• Store aliquots at -20°C or -80°C under sterile conditions. For short-term use (up to 1 month), storage at 2–8°C is acceptable.
• If desired, add 0.1% bovine serum albumin (BSA) to the PBS during reconstitution to stabilize the protein, especially for long-term storage or low concentration working solutions.
• Avoid diluting below 100 μg/mL for storage to maintain protein stability.

Preparation for cell culture experiments:

• Before use, dilute the reconstituted stock to the desired working concentration using sterile cell culture medium or PBS. Typical bioactive concentrations for IL-12 in cell assays range from 0.04 ng/mL to 1 ng/mL, depending on cell type and experimental design.
• Filter the final working solution through a 0.2 μm sterile filter if sterility is critical.
• Confirm bioactivity using a suitable assay, such as IFN-γ induction in responsive cell lines (e.g., NK-92MI).

Summary Table:

This protocol ensures optimal solubility, stability, and bioactivity of recombinant human IL-12 for cell culture applications.