Recombinant Human IL-2Rα

Recombinant Human IL-2Rα (CD25) is used in research applications to study and modulate immune responses, particularly T cell activation, regulation, and competition for IL-2, as well as to investigate mechanisms relevant to cancer, autoimmunity, and inflammation.

Key scientific applications and rationale:

• Competitive Regulation of IL-2 Signaling: IL-2Rα is the high-affinity component of the IL-2 receptor complex, enabling T cells to efficiently capture and respond to IL-2, especially in environments where IL-2 is limited. Recombinant IL-2Rα can be used to dissect how T cells compete for IL-2, which is critical for understanding effector and regulatory T cell dynamics in cancer and infectious disease models.

• Soluble IL-2Rα as a Biomarker and Functional Modulator: Soluble forms of IL-2Rα (sIL-2Rα) are found in biological fluids and correlate with immune activation and certain pathologies, such as leukemias, lymphomas, and inflammatory conditions. Recombinant sIL-2Rα can be used to study its role as a biomarker and its ability to inhibit IL-2-dependent cell proliferation, providing insights into immune regulation and disease progression.

• Mechanistic Studies of T Cell Differentiation and Exhaustion: IL-2Rα-mediated signaling is crucial for the differentiation of antigen-specific CD8+ T cells into effective cytotoxic cells and for overcoming T cell exhaustion, especially in chronic infection and cancer. Recombinant IL-2Rα allows researchers to manipulate and analyze these pathways in vitro and in vivo.

• Therapeutic Development and Screening: Recombinant IL-2Rα is valuable for screening and characterizing IL-2 variants, antibodies, and fusion proteins designed to selectively target or modulate the IL-2/IL-2Rα axis for immunotherapy, including cancer and autoimmune disease models.

Best practices and protocols:

• Use recombinant IL-2Rα in binding assays to quantify IL-2 affinity and receptor-ligand interactions.
• Employ recombinant sIL-2Rα in cell culture to modulate IL-2 availability and study its effects on T cell proliferation and differentiation.
• Analyze serum or culture supernatants for sIL-2Rα as a readout of immune activation or disease state.

Summary of scientific value:

• Recombinant Human IL-2Rα is a critical tool for dissecting IL-2 signaling, immune cell competition, and regulatory mechanisms, with direct relevance to immunotherapy, biomarker discovery, and basic immunology research.

You can use recombinant human IL-2Rα as a standard for quantification or calibration in ELISA assays, provided that the ELISA kit is validated to recognize both recombinant and natural forms of IL-2Rα and that the recombinant standard is of high purity and correctly quantified.

Key considerations and supporting details:

• ELISA Kit Validation: Many commercial ELISA kits for human IL-2Rα are specifically designed to use recombinant human IL-2Rα as the standard. These kits typically generate a standard curve using serial dilutions of recombinant IL-2Rα, which is then used to interpolate concentrations in unknown samples.
• Parallelism: Well-validated kits demonstrate that the standard curves generated with recombinant IL-2Rα are parallel to those generated with natural IL-2Rα, indicating that the assay quantifies both forms equivalently.
• Specificity: Most sandwich ELISA kits for IL-2Rα are highly specific and do not show significant cross-reactivity with related proteins or analogues, ensuring accurate quantification.
• Standard Preparation: The recombinant IL-2Rα standard should be reconstituted and diluted according to the kit’s instructions to ensure accurate calibration.
• Research Use: These standards and kits are typically for research use only and not for diagnostic purposes.

Best Practices:

• Always use a recombinant standard that matches the sequence and post-translational modifications (e.g., glycosylation) of the native protein as closely as possible.
• Confirm that your ELISA kit documentation explicitly states compatibility with recombinant IL-2Rα as a standard.
• Prepare the standard curve in the same matrix as your samples (e.g., serum, plasma, or buffer) to minimize matrix effects.

Limitations:

• If your recombinant IL-2Rα differs significantly in structure or glycosylation from the native protein in your samples, minor quantification discrepancies may occur, but most commercial kits are designed to minimize this issue.
• Always check the kit’s technical documentation for any specific recommendations or limitations regarding the use of recombinant standards.

In summary, recombinant human IL-2Rα is widely accepted and routinely used as a standard for ELISA quantification, provided the assay is validated for this purpose and the standard is prepared according to protocol.

Recombinant Human IL-2Rα (CD25) has been validated in published research for several key applications, primarily in immunological assays and cell biology studies. The most commonly reported applications include:

• ELISA (Enzyme-Linked Immunosorbent Assay): Recombinant IL-2Rα is widely used as a standard or capture reagent in ELISA to quantify soluble IL-2Rα in biological fluids, which serves as a biomarker for immune activation, inflammation, and certain malignancies.
• Bioassays: It is used in cell-based bioassays to assess its ability to inhibit IL-2-dependent cell proliferation, such as in MO7e human megakaryocytic leukemic cells.
• Protein-Protein Interaction Studies: Recombinant IL-2Rα is employed to study interactions with IL-2 and other receptor subunits, as well as to characterize binding kinetics and affinity in vitro.
• Immunological Research: It is used to investigate the role of IL-2Rα in T cell and B cell activation, immune regulation, and disease pathogenesis.
• Therapeutic Development: Recombinant IL-2Rα is utilized in the development and characterization of therapeutic antibodies targeting CD25 (e.g., daclizumab) and in the engineering of fusion proteins or immunocytokines for cancer immunotherapy.

Supporting details and context:

• ELISA and Immunoassays: Multiple studies and product datasheets confirm the use of recombinant IL-2Rα as an ELISA standard or capture reagent to measure soluble IL-2Rα in serum or plasma, which correlates with immune activation in conditions such as leukemia, lymphoma, and autoimmune diseases.
• Bioactivity Assays: Its activity is validated by its ability to inhibit IL-2-driven proliferation in specific cell lines, providing a functional readout for receptor-ligand interactions.
• Therapeutic Antibody Characterization: Recombinant IL-2Rα is used to assess the binding and functional properties of anti-CD25 antibodies, including glycosylation analysis and antibody-dependent cell-mediated cytotoxicity.
• Fusion Protein and Immunocytokine Research: Engineered molecules combining IL-2 or IL-2Rα domains are tested for selective immune cell activation and anti-tumor efficacy in preclinical models.

Additional applications:

• Cell Culture: Recombinant IL-2Rα may be used as a supplement or experimental variable in cell culture systems to study T cell biology and cytokine signaling.
• Protein Interaction and Structural Studies: It is used in biophysical assays (e.g., SPR, BLI) and crystallography to elucidate the structure and binding properties of the IL-2/IL-2R complex.

Summary Table of Validated Applications

These applications are well-supported in the literature and by product validation data from multiple research-focused sources.

To reconstitute and prepare Recombinant Human IL-2Rα (CD25) protein for cell culture experiments, dissolve the lyophilized protein in sterile PBS containing at least 0.1% human or bovine serum albumin (BSA) to a final concentration of 50–100 μg/mL. This approach helps maintain protein stability and prevents adsorption to plasticware.

Step-by-step protocol:

• Briefly centrifuge the vial to collect the lyophilized powder at the bottom before opening.
• Add sterile PBS (phosphate-buffered saline) containing at least 0.1% BSA or human serum albumin to achieve the desired concentration (typically 50–100 μg/mL).
• Gently mix by pipetting up and down or by slow inversion. Avoid vigorous shaking or vortexing to prevent protein denaturation.
• Allow the protein to fully dissolve (usually within a few minutes).
• Aliquot the reconstituted protein into sterile tubes to avoid repeated freeze-thaw cycles.
• Store aliquots at 2–8 °C for up to 1 month or at –20 °C to –70 °C for up to 3 months under sterile conditions.

Additional notes for cell culture use:

• For further dilution into cell culture medium, use low endotoxin buffers and supplement with FBS or tissue culture grade BSA if needed.
• Always work under sterile conditions to prevent contamination.
• Avoid repeated freeze-thaw cycles, as this can reduce protein activity and stability.
• If the protein is carrier-free, reconstitution in sterile PBS alone is acceptable, but adding BSA or human serum albumin is recommended for stability, especially at low concentrations.

Summary Table:

This protocol ensures optimal solubility, stability, and biological activity of recombinant IL-2Rα for cell culture experiments.