Recombinant Human IL-4 Rα

Recombinant Human IL-4 Rα is widely used in research applications to study and modulate Th2-biased immune responses, allergic inflammation, and cytokine signaling, as it is the primary receptor for both IL-4 and IL-13, key mediators in type 2 immunity.

Key scientific applications and rationale:

• Mechanistic Studies: Recombinant IL-4 Rα enables detailed investigation of IL-4/IL-13 signaling pathways, which are central to the differentiation of naïve CD4+ T cells into Th2 cells, alternative macrophage activation, and regulation of mucosal immunity. This is crucial for understanding the molecular basis of allergic diseases, asthma, and atopic dermatitis.

• Antibody Screening and Validation: It serves as a standard for screening and release assays for monoclonal antibodies or engineered proteins that block IL-4 signaling, such as those targeting allergic and autoimmune diseases. For example, antagonistic antibodies against IL-4Rα can be tested for their ability to inhibit IL-4/IL-13-induced cell proliferation and STAT6 activation.

• Disease Modeling: Recombinant IL-4 Rα is used in preclinical models to evaluate therapeutic candidates for type 2 inflammatory diseases, including asthma, atopic dermatitis, and allergic rhinitis. Its role in modulating immune responses makes it a valuable tool for dissecting disease mechanisms and testing interventions.

• Genetic and Functional Studies: It allows for the exploration of genetic variations in IL-4Rα that impact susceptibility to autoimmune diseases, viral infections, and cancer progression. Recombinant protein can be used to study receptor-ligand interactions, downstream signaling, and the effects of specific mutations.

• Cancer Immunology: IL-4Rα is implicated in tumor cell proliferation and metastasis, particularly in hepatocellular carcinoma, by modulating JAK1/STAT6 and JNK/ERK1/2 pathways. Recombinant IL-4 Rα can be used to investigate these processes and develop targeted therapies.

Best practices:

• Use recombinant IL-4 Rα in in vitro binding assays (e.g., SPR, ELISA) to quantify affinity and blocking efficacy of candidate molecules.
• Employ it in cell-based assays to assess functional outcomes such as cytokine-induced proliferation, differentiation, and activation.
• Integrate recombinant IL-4 Rα in animal models expressing humanized receptors to evaluate therapeutic efficacy and pharmacokinetics.

Summary of advantages:

• Specificity: Enables precise targeting and modulation of IL-4/IL-13 signaling.
• Versatility: Applicable in immunology, allergy, autoimmunity, and oncology research.
• Translational relevance: Facilitates development and validation of novel therapeutics for type 2 inflammatory and immune-mediated diseases.

In conclusion, recombinant human IL-4 Rα is an essential reagent for dissecting cytokine signaling, validating therapeutic candidates, and modeling immune-related diseases, making it highly valuable for both basic and translational research.

Recombinant Human IL-4 Rα can be used as a standard for quantification or calibration in ELISA assays, but only if the ELISA is specifically designed to detect IL-4 Rα and the recombinant protein is validated for use as a standard in that assay. The suitability depends on the assay format, antibody specificity, and validation data.

Key considerations:

• Assay specificity: ELISA kits for IL-4 Rα quantification typically use recombinant human IL-4 Rα as a standard, provided the antibodies in the kit recognize both recombinant and native forms equivalently. The standard must be well-characterized, with known concentration and purity, and ideally matched to the protein detected in your samples.

• Validation: The recombinant standard should be validated for parallelism with native IL-4 Rα in your sample matrix. This ensures that the standard curve accurately reflects the analyte in biological samples. Commercial ELISA kits for IL-4 Rα often report such validation, showing parallel standard curves for recombinant and natural protein.

• Purity and endotoxin: High purity (>95%) and low endotoxin levels (<1 EU/µg) are important for reproducibility and to avoid interference in sensitive assays.

• Intended use: Not all recombinant proteins are validated for use as ELISA standards. Some are intended for bioassays or Western blot only. Always check the product documentation for recommended applications.

• Calibration: Prepare a dilution series of the recombinant IL-4 Rα in the same buffer as your samples to generate a standard curve. Use this curve to interpolate concentrations in unknown samples, ensuring the assay’s dynamic range and sensitivity are appropriate for your needs.

Limitations:

• If your ELISA is designed for IL-4 (the cytokine) rather than IL-4 Rα (the receptor), you cannot use IL-4 Rα as a standard; the antibodies will not recognize it.
• If the recombinant IL-4 Rα is not validated for ELISA standard use, you must perform your own validation (e.g., parallelism, recovery, accuracy) before relying on it for quantification.

Best practices:

• Use recombinant IL-4 Rα only in ELISA assays specifically designed for IL-4 Rα quantification.
• Confirm that your recombinant standard is validated for ELISA calibration, either by the supplier or through your own assay validation.
• Always run a fresh standard curve with each assay to ensure accuracy.

In summary, recombinant human IL-4 Rα is suitable as a standard for ELISA quantification only in validated IL-4 Rα assays. It cannot be used for IL-4 cytokine quantification. Always verify assay compatibility and perform necessary validation steps for reliable results.

Recombinant Human IL-4 Rα has been validated for several key applications in published research, primarily in the context of immunology and inflammation studies. The most common validated applications include:

• In vitro binding and blocking assays: Recombinant human IL-4 Rα is used to assess the binding affinity and blocking efficacy of monoclonal antibodies or engineered antagonists targeting IL-4Rα, often via surface plasmon resonance (SPR) and ELISA-based assays.
• Cell-based functional assays: It is employed to evaluate inhibition of IL-4- and IL-13-mediated signaling pathways, such as STAT6 activation, and to measure effects on cell proliferation (e.g., TF-1 cell proliferation assays).
• T cell differentiation studies: Recombinant IL-4 Rα is used to investigate its role in blocking the differentiation of naïve CD4+ T cells into Th2 cells, which is central to allergic and type 2 inflammatory responses.
• Preclinical in vivo models: Transgenic mice expressing human IL-4Rα are used to validate therapeutic antibodies or antagonists in models of asthma, atopic dermatitis, and allergic rhinitis, with endpoints including airway hyperresponsiveness, serum IgE levels, and tissue inflammation.
• Bioassays and standards: It serves as a standard or control in bioassays for quantifying IL-4 or IL-13 activity and for screening inhibitory molecules that target IL-4 signaling.

Supporting details and examples:

• In the development of monoclonal antibodies such as SHR-1819 and CM310, recombinant human IL-4Rα was used in ELISA and SPR assays to determine binding kinetics and blocking potency.
• Functional assays using recombinant IL-4Rα demonstrated inhibition of IL-4/IL-13-induced STAT6 activation and TF-1 cell proliferation, both standard readouts for IL-4Rα activity.
• In ex vivo studies, recombinant IL-4Rα was used to block IL-4-driven Th2 differentiation from human peripheral blood mononuclear cells, a critical step in validating antagonistic antibodies.
• In vivo, human IL-4Rα transgenic mice enabled the evaluation of therapeutic efficacy in disease models, confirming the receptor’s role in mediating type 2 inflammation.

Additional relevant information:

• Recombinant human IL-4Rα is also used in the development and validation of enzyme-linked immunosorbent assays (ELISAs) for pharmacokinetic studies of anti-IL-4Rα antibodies.
• It is a key reagent for screening and release assays for both antibodies and engineered IL-4 variants, supporting drug discovery and development pipelines.

In summary, recombinant human IL-4 Rα is a validated tool for a broad range of applications in immunological research, especially for studying and targeting type 2 inflammatory pathways.

To reconstitute and prepare Recombinant Human IL-4 Rα protein for cell culture experiments, dissolve the lyophilized protein in sterile PBS containing at least 0.1% human or bovine serum albumin (HSA or BSA) to a final concentration of 100 μg/mL. This carrier protein is essential for stabilizing the recombinant protein and minimizing adsorption to surfaces.

Step-by-step protocol:

• Briefly centrifuge the vial before opening to collect all lyophilized material at the bottom.
• Add sterile PBS (pH 7.2–7.4) containing ≥0.1% HSA or BSA to achieve the desired concentration (typically 100 μg/mL).
• Gently mix by swirling or tapping; do not vortex or shake vigorously to avoid denaturation or foaming.
• Ensure complete dissolution by allowing the vial to sit at room temperature for a few minutes if necessary.
• Aliquot the reconstituted protein to avoid repeated freeze-thaw cycles, which can degrade activity.
• 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 several months.
• Dilution for cell culture:
  • Further dilute the stock solution in cell culture medium immediately before use.
  • Always include a carrier protein (e.g., 0.1–1% HSA or BSA) in all working solutions to maintain stability.

Additional best practices:

• Use aseptic technique throughout to prevent contamination.
• Avoid concentrations above 1 mg/mL during reconstitution to prevent solubility issues.
• If the protein is supplied carrier-free, always add carrier protein during reconstitution and dilution steps.
• Confirm protein concentration and integrity by SDS-PAGE if needed.

This protocol ensures optimal solubility, stability, and bioactivity of recombinant IL-4 Rα for cell culture applications.