Recombinant Human IL-12/IL-23 p40

Recombinant Human IL-12/IL-23 p40 is used in research applications to investigate immune regulation, autoimmune disease mechanisms, and cytokine signaling due to its unique biological activities distinct from the full IL-12 and IL-23 heterodimers.

Key scientific reasons to use recombinant human IL-12/IL-23 p40 include:

• Modulation of Immune Responses: The p40 subunit, as a monomer or homodimer, can inhibit IL-12 and IL-23 signaling by competing for receptor binding, thereby regulating Th1 and Th17 cell responses. This property is crucial for dissecting cytokine signaling pathways and understanding immune cell differentiation.

• Anti-Autoimmune Effects: Recombinant p40 has demonstrated the ability to suppress clinical symptoms and disease progression in experimental autoimmune encephalomyelitis (EAE) and collagen-induced arthritis models, acting as an anti-autoimmune cytokine by attenuating IL-12Rβ1 internalization and protecting regulatory T cells. This makes it valuable for studying autoimmune disease mechanisms and potential therapeutic interventions.

• Functional Assays: Recombinant p40 enhances IFN-γ secretion in NK-92 human natural killer cells, making it useful for functional assays involving NK cell activation and cytokine profiling. The ED50 for this effect is reported to be 2.00–20.0 ng/mL.

• Inhibition of IL-23 Functions: The secreted p40 subunit can inhibit IL-23-mediated effects, including anti-tumor responses, which is relevant for cancer immunology studies.

• Tool for ELISA and Quantification: Recombinant p40 is used as a standard in ELISA kits for quantifying IL-12/IL-23 p40 levels in biological samples, supporting biomarker studies and cytokine profiling.

• Sequence Conservation: Human p40 shares significant sequence identity with mouse and rat p40, facilitating cross-species studies and translational research.

• Mechanistic Studies: The availability of recombinant p40 allows for precise mechanistic studies, such as investigating receptor interactions, cytokine competition, and downstream signaling events.

In summary, recombinant human IL-12/IL-23 p40 is a versatile reagent for immunology research, enabling detailed analysis of cytokine signaling, immune regulation, and disease mechanisms, especially in contexts where modulation of IL-12 and IL-23 pathways is relevant.

Yes, recombinant human IL-12/IL-23 p40 can be used as a standard for quantification and calibration in ELISA assays. This is a well-established practice in immunoassay development and validation.

Calibration and Standardization

Recombinant IL-12/IL-23 p40 serves as the primary calibration standard in commercial ELISA kits designed to measure this cytokine. The recombinant protein is typically expressed in insect cell systems and highly purified to ensure consistency and reproducibility across assays. When used as a calibration standard, the recombinant p40 produces dose-response curves that are parallel to those obtained with natural human IL-12/IL-23 p40, indicating that the recombinant protein accurately represents the native form for quantification purposes.

Accuracy and Validation

The recombinant standard demonstrates excellent accuracy for quantifying both recombinant and natural forms of IL-12/IL-23 p40. This dual recognition capability is critical because it allows a single standard curve to be used for measuring the cytokine from various biological sources, including cell culture supernatants, serum, and plasma samples. The parallel dose-response curves between recombinant and natural IL-12/IL-23 p40 validate that relative mass values can be reliably determined using the recombinant standard.

Binding Characteristics

The recombinant p40 subunit demonstrates specific binding interactions with IL-12 receptor components, with binding affinity (ED₅₀) values in the range of 1-6 ng/mL when immobilized IL-12 receptor beta 1 is used as the capture molecule. This binding specificity ensures that the standard behaves appropriately in sandwich immunoassay formats.

Practical Considerations

When implementing recombinant IL-12/IL-23 p40 as your calibration standard, ensure that you reconstitute it according to manufacturer specifications using appropriate diluents (typically deionized or distilled water), prepare serial dilutions with the correct calibrator diluent matched to your sample type, and maintain consistency in your dilution series to generate accurate standard curves.

Recombinant Human IL-12/IL-23 p40 has been validated for several key applications in published research, primarily in immunological assays and functional studies.

Validated Applications:

• Bioassays: Used to assess cytokine activity, including stimulation of cytotoxic activity, proliferation, and promotion of Th1 development in T cells and NK cells. It has been employed to measure IFN-γ secretion in NK-92 cells and to study the inhibition of IL-23-mediated anti-tumor effects.
• ELISA (Enzyme-Linked Immunosorbent Assay): Frequently used as a standard or analyte for quantifying IL-12/IL-23 p40 in serum, plasma, and cell culture supernatants. This includes specificity testing and quantitative detection in various sample types.
• Antibody Validation: Utilized in the validation of antibodies targeting the p40 subunit for applications such as flow cytometry (intracellular staining) and ELISA.
• Functional Blocking and Inhibition Studies: Applied in research investigating the inhibition of IL-12 or IL-23 binding to their receptors, and in studies of immune modulation, such as blocking Th1/Th17 responses or anti-tumor immunity.
• Cellular and Molecular Immunology: Used to study cytokine signaling, receptor binding, and immune cell differentiation, particularly in the context of autoimmune diseases, allergy, asthma, and cancer.

Supporting Details:

• Bioassay Examples: Recombinant Human IL-12/IL-23 p40 has been shown to bind IL-12Rβ1 and induce IFN-γ secretion in NK cells, confirming its biological activity.
• ELISA Validation: Multiple commercial and academic studies have validated its use as a standard for ELISA kits, enabling accurate quantification of IL-12/IL-23 p40 in biological samples.
• Antibody Validation: Recombinant p40 is used to confirm antibody specificity and reactivity in both ELISA and flow cytometry protocols.
• Therapeutic Research: Recombinant p40 has been used in the development and validation of vaccines and therapeutic antibodies targeting autoimmune and inflammatory diseases, such as Crohn’s disease, multiple sclerosis, and rheumatoid arthritis.

Summary Table:

These applications are well-supported in the literature and are foundational for immunological research involving IL-12 and IL-23 signaling pathways.

To reconstitute and prepare Recombinant Human IL-12/IL-23 p40 protein for cell culture experiments, dissolve the lyophilized protein in sterile phosphate-buffered saline (PBS) to a concentration of 100 μg/mL. Use gentle mixing and avoid vortexing to preserve protein integrity.

Step-by-step protocol:

• Reconstitution:

  • Add sterile PBS directly to the lyophilized protein to achieve the desired concentration (commonly 100 μg/mL).
  • If the protein appears as a film, gently swirl or tap the vial to ensure complete dissolution.
  • For extended storage, consider adding 0.1% bovine serum albumin (BSA) to stabilize the protein.

• Mixing:

  • Mix gently by inversion or slow pipetting. Do not vortex, as this may denature the protein.

• Aliquoting and Storage:

  • Aliquot the reconstituted protein into sterile polypropylene tubes to avoid repeated freeze-thaw cycles.
  • Store aliquots at −20 °C to −70 °C for long-term use, or at 2–8 °C for short-term use (up to 1 month).
  • Avoid repeated freeze-thaw cycles to maintain bioactivity.

• Preparation for Cell Culture:

  • Before use, dilute the stock solution to the working concentration required for your experiment using sterile cell culture medium or PBS.
  • Typical working concentrations for functional assays range from 2–20 ng/mL, depending on cell type and experimental design.

• Sterility:

  • Ensure all solutions and containers are sterile to prevent contamination of cell cultures.

Additional notes:

• If the protein is supplied with a carrier (e.g., BSA or trehalose), this can help stabilize the protein during reconstitution and storage.
• Always check the endotoxin level if using for sensitive cell culture applications; recombinant proteins for cell culture should have low endotoxin (<0.1 EU/μg).
• For best results, prepare fresh working solutions immediately before use and avoid storing diluted protein for extended periods.

This protocol ensures optimal solubility, stability, and biological activity of recombinant IL-12/IL-23 p40 for cell culture experiments.