Recombinant Mouse IL-12 p40 (Homodimer)

Recombinant Mouse IL-12 p40 (Homodimer) is primarily used in research as a potent antagonist of IL-12 activity, enabling the study of IL-12-dependent immune responses and the modulation of inflammatory and autoimmune processes. It is especially valuable for dissecting the roles of IL-12 and IL-23 in disease models and for investigating the mechanisms of immune regulation.

Key scientific applications and rationale include:

• IL-12 Antagonism: The p40 homodimer binds to the IL-12 receptor but does not trigger signaling, thereby competitively inhibiting the activity of native IL-12 (p70) and IL-23. This allows researchers to specifically block IL-12/IL-23 pathways in vitro and in vivo, clarifying their roles in immune cell activation, cytokine production, and disease progression.

• Potency Compared to Monomer: The homodimeric form is 25–50 times more active than the p40 monomer as an IL-12 antagonist, making it the preferred reagent for functional inhibition studies.

• Autoimmune and Inflammatory Disease Models: Administration of recombinant p40 homodimer has been shown to ameliorate disease severity in mouse models of experimental autoimmune encephalomyelitis (EAE, a model for multiple sclerosis) and collagen-induced arthritis (CIA, a model for rheumatoid arthritis). This demonstrates its utility in probing the contribution of IL-12/IL-23 to autoimmune pathogenesis and in testing therapeutic strategies.

• Mechanistic Studies: By blocking IL-12/IL-23 signaling, the p40 homodimer enables detailed analysis of downstream effects, such as changes in T cell responses (Th1/Th17), cytokine profiles (e.g., IFN-γ, IL-17, TNF-α), and barrier integrity in the central nervous system.

• Bioassays and Receptor Studies: The p40 homodimer is used in bioassays to confirm IL-12 receptor specificity and to distinguish between receptor binding and signaling functions.

• Tool for Immunomodulation: Its ability to modulate immune responses makes it a valuable tool for studying the balance between pro- and anti-inflammatory signals in various experimental systems.

Summary of best practices:

• Use the homodimeric form for maximal antagonistic effect.
• Employ in both in vitro and in vivo models to dissect IL-12/IL-23 function.
• Combine with disease models to evaluate therapeutic potential and mechanistic pathways.

In summary, recombinant mouse IL-12 p40 homodimer is a critical reagent for immunological research, particularly for investigating and modulating IL-12/IL-23-driven processes in autoimmunity, inflammation, and immune regulation.

Yes, recombinant mouse IL-12 p40 homodimer can be used as a standard for quantification and calibration in ELISA assays, though there are important considerations regarding specificity and experimental design.

Suitability as an ELISA Standard

The recombinant p40 homodimer is specifically designed for use as a quantitative standard in sandwich ELISA formats. It is particularly useful for measuring IL-12/IL-23 p40 protein levels, as the antibodies in these assay systems recognize the p40 subunit in multiple forms—including the monomer, homodimer, and as part of the IL-12 p70 heterodimer.

Formulation Considerations

When using recombinant p40 homodimer as a standard, the formulation choice is critical. The protein is available in two formats: with carrier protein (typically bovine serum albumin) or carrier-free. For ELISA standard applications, the carrier protein-containing formulation is generally recommended. If you select the carrier-free version, ensure that carrier proteins at concentrations of 5-10 mg/mL are added to prevent loss of activity during storage and handling.

Important Precautions

Storage and stability: Failure to add appropriate carrier protein or maintain proper storage temperatures may result in significant loss of activity. Carrier proteins should be pre-screened for potential interference in your specific experimental system, as they may affect results through toxicity, high endotoxin levels, or blocking activity.

Cross-reactivity specificity: When using p40 homodimer standards, verify that your detection antibodies do not cross-react with other cytokines. For instance, some assay systems show no cross-reactivity with IL-12 p70 or IL-23, while others are designed to detect p40 across multiple IL-12/IL-23 contexts.

Recommended Dilution Range

Standard curves typically employ doubling dilutions ranging from approximately 4,000 to 30 pg/mL, depending on your assay sensitivity requirements. Typical assay sensitivities for p40-specific ELISAs range from 3.7 to 5 pg/mL.

Strain-Specific Considerations

If working with specific mouse strains, note that polymorphisms exist in the mouse IL-12/IL-23 p40 sequence. Some capture antibodies are allele-specific and recognize only certain p40 variants, while others recognize both variant and non-variant forms. Verify antibody compatibility with your recombinant standard.

Recombinant Mouse IL-12 p40 (Homodimer) has been validated in published research primarily as an IL-12 antagonist in both in vitro and in vivo applications, as well as for its potential agonist activities in certain immune contexts.

Key validated applications include:

• Inhibition of IL-12-dependent responses in vitro:
  The p40 homodimer has been shown to potently inhibit IL-12-induced proliferation of mouse splenocytes, IFN-γ secretion, and NK cell activation in cell-based assays. This is typically measured by bioassays assessing cytokine production or cell proliferation.

• In vivo studies as an IL-12 antagonist:
  The p40 homodimer has been used to block IL-12 activity in animal models, allowing researchers to dissect the role of IL-12 in immune responses, inflammation, and disease models such as autoimmunity and infection.

• ELISA standard:
  It has been used as a standard in ELISA assays to quantify IL-12 p40 levels in biological samples, particularly in studies of inflammation and cytokine profiling.

• Western blot:
  The protein has been validated for use in Western blotting to detect IL-12 p40 in cell lysates or tissue samples.

• Bioassays for functional studies:
  The homodimer is widely used in bioassays to study its effects on immune cell signaling, cytokine production, and as a tool to probe IL-12 receptor interactions.

• Agonist activities:
  Some studies report that the p40 homodimer can act as a chemoattractant for macrophages and dendritic cells, induce iNOS expression in microglia, and modulate leukocyte accumulation, suggesting additional pro-inflammatory roles beyond antagonism.

• In vivo models of disease:
  The protein has been used in models of pulmonary fibrosis, neuroinflammation, and airway inflammation to study its role in disease pathogenesis and immune regulation.

Summary Table of Validated Applications

Additional Notes:

• The p40 homodimer is significantly more potent than the monomer as an IL-12 antagonist.
• Its physiological role in vivo is still under investigation, but it is a well-established tool for dissecting IL-12 biology in experimental systems.
• Some studies have used it to explore its effects on Th1/Th17 responses, nitric oxide production, and neuroimmune interactions.

If you need protocol details or specific experimental setups for any of these applications, please specify the context or assay type.

To reconstitute and prepare Recombinant Mouse IL-12 p40 (Homodimer) for cell culture experiments, dissolve the lyophilized protein at 100 μg/mL in sterile PBS containing at least 0.1% human or bovine serum albumin (BSA or HSA) to enhance stability and prevent adsorption to surfaces.

Step-by-step protocol:

• Centrifuge the vial briefly before opening to ensure all material is at the bottom.
• Add sterile PBS (phosphate-buffered saline) containing at least 0.1% BSA or HSA to the vial to achieve a final concentration of 100 μg/mL.
• Gently pipette up and down to fully dissolve the protein, washing down the sides of the vial to recover all material.
• Allow the solution to sit for a few minutes at room temperature to ensure complete dissolution.
• Aliquot the reconstituted protein to avoid repeated freeze-thaw cycles, which can degrade the protein.
• Storage:
  • Short-term: Store aliquots at 2–8°C for up to one week.
  • Long-term: Store at –20°C or colder for up to 3 months.
• Avoid repeated freeze-thaw cycles to maintain protein integrity.

Dilution for cell culture:

• Before adding to cell cultures, further dilute the stock solution in cell culture medium to the desired working concentration, ensuring the final buffer composition is compatible with your cells.
• If using serum-free media, maintain a low concentration of carrier protein (e.g., 0.1% BSA) to prevent protein loss due to adsorption.

Additional notes:

• If the product is supplied carrier-free, adding 0.1–1% BSA or HSA during reconstitution is strongly recommended to stabilize the protein.
• Always use sterile technique to prevent contamination.
• The biological activity of the IL-12 p40 homodimer should be validated in your specific assay system, as activity may vary depending on the application.

This protocol is suitable for preparing recombinant mouse IL-12 p40 homodimer for use in cell culture experiments, including functional assays and cytokine stimulation studies.