Recombinant Human Protein CTLA-4 (CD152)

Recombinant Human Protein CTLA-4 (CD152) is a critical tool for research applications focused on immune regulation, immunotherapy, and T-cell biology. It enables precise investigation of immune checkpoint mechanisms, therapeutic development, and functional assays relevant to cancer, autoimmunity, and tolerance.

Key scientific reasons to use recombinant human CTLA-4 in research include:

• Immune Checkpoint Studies: CTLA-4 is a major inhibitory receptor on T cells, acting as a negative regulator of immune activation. Recombinant CTLA-4 allows researchers to dissect its role in downregulating T-cell responses and maintaining immune homeostasis, especially by competing with CD28 for binding to CD80/CD86 on antigen-presenting cells.

• Cancer Immunotherapy Research: CTLA-4 blockade is a validated strategy in cancer immunotherapy, as demonstrated by clinical success with checkpoint inhibitors. Recombinant CTLA-4 is essential for in vitro neutralization assays, mechanistic studies, and the development of novel therapeutic agents targeting this pathway.

• Autoimmunity and Tolerance: Augmenting CTLA-4 signaling can suppress excessive immune responses, making recombinant CTLA-4 valuable for modeling and understanding autoimmune diseases and tolerance induction.

• Functional Assays and Screening: Recombinant CTLA-4 is used in binding assays, flow cytometry, and ELISA to quantify ligand interactions, screen for inhibitors, and validate antibody specificity.

• Protein Engineering and Therapeutic Development: Recombinant CTLA-4 serves as a template for designing fusion proteins, nanobodies, and engineered receptors for therapeutic applications, including immune modulation and targeted delivery.

• Modeling T-cell Regulation: CTLA-4-deficient models demonstrate the protein’s essential role in preventing lymphoproliferative disease and autoimmunity, highlighting its importance in basic and translational research.

Summary of applications:

• Mechanistic studies of immune checkpoints
• Development and validation of immunotherapeutics
• Functional assays (neutralization, binding, flow cytometry)
• Autoimmunity and tolerance research
• Protein engineering and drug screening

Using recombinant human CTLA-4 provides a standardized, reproducible reagent for these applications, facilitating high-quality, translational research in immunology and related fields.

Yes, recombinant human CTLA-4 (CD152) protein can be used as a standard for quantification and calibration in ELISA assays. This is a well-established application supported by multiple validated protocols and quality-controlled products.

Suitability for ELISA Standards

Recombinant human CTLA-4 protein is specifically designed and validated for use as an ELISA standard. The protein is quality control tested by ELISA assay for each production lot, ensuring consistency and reliability across experiments. These standards are guaranteed suitable for sandwich ELISA applications, which is the most common format for CTLA-4 quantification.

Key Specifications for Calibration

When using recombinant CTLA-4 as a standard, consider these important characteristics:

Protein Properties: Recombinant CTLA-4 proteins are typically expressed in mammalian systems (such as human 293 cells or CHO cells) and contain affinity tags (His tag, Twin-Strep tag, or Fc tag) for purification and detection. The protein exhibits high purity, generally exceeding 85-95% as determined by SDS-PAGE.

Molecular Weight Considerations: The calculated molecular weight of CTLA-4 is approximately 16.5 kDa, though the protein may migrate at 20-30 kDa under reducing conditions due to glycosylation. This is important to account for when preparing your standard curve.

Binding Characteristics: Recombinant CTLA-4 protein demonstrates specific binding to B7-2 (CD86) and can be captured by anti-CTLA-4 antibodies with defined linear ranges (typically 1-20 ng/mL depending on the specific protein variant).

Practical Considerations for Standard Preparation

When reconstituting lyophilized recombinant CTLA-4 standards, follow the manufacturer's protocol carefully. Standards are typically reconstituted in appropriate dilution buffers to achieve a stock concentration (commonly 10 ng/mL). Use a fresh standard for each assay to ensure accuracy.

The assay range for human CTLA-4 detection typically spans from approximately 23.4 pg/mL to 1500 pg/mL, depending on the specific kit formulation, providing adequate dynamic range for most quantification applications.

Recombinant Human Protein CTLA-4 (CD152) has been validated for a range of applications in published research, primarily in the fields of immunology, cancer immunotherapy, and autoimmune disease studies.

Key validated applications include:

• Immune checkpoint research: Recombinant CTLA-4 is widely used to study immune regulation, specifically the inhibition of T-cell activation and the mechanisms of immune tolerance.
• Cancer immunotherapy studies: It is used to investigate immune checkpoint blockade strategies, including the development and testing of monoclonal antibodies (e.g., ipilimumab) and fusion proteins (e.g., CTLA-4-Ig) for enhancing anti-tumor immune responses.
• Autoimmune disease models: Recombinant CTLA-4 and its fusion proteins have been applied in preclinical and clinical studies for autoimmune conditions such as rheumatoid arthritis, where modulation of T-cell activity is therapeutically relevant.
• Quantification and detection of CTLA-4 expression: The protein is used as a standard or antigen in assays such as flow cytometry, ELISA, and immunofluorescence to quantify CTLA-4-positive lymphocytes and analyze CTLA-4 expression on T cells.
• In vitro functional assays: Recombinant CTLA-4 is employed in in vitro neutralization assays to study its inhibitory effects on T-cell activation and to test the efficacy of blocking antibodies or ligands.
• Development of therapeutic fusion proteins: CTLA-4-Ig fusion proteins, based on recombinant CTLA-4, are used in both research and clinical settings to inhibit co-stimulatory signals and modulate immune responses.
• Structural and biochemical studies: Recombinant CTLA-4 is used for protein-protein interaction studies, ligand binding assays (with CD80/CD86), and structural characterization.

Summary Table of Validated Applications

These applications are supported by a substantial body of published research and ongoing clinical trials, reflecting the central role of recombinant CTLA-4 in both basic and translational immunology.

To reconstitute and prepare Recombinant Human Protein CTLA-4 (CD152) for cell culture experiments, first centrifuge the vial briefly to collect the lyophilized powder at the bottom. Add sterile distilled water to achieve a final concentration typically between 0.1–1.0 mg/mL; a common working concentration is 0.2–0.5 mg/mL. Avoid vigorous mixing—gently swirl or invert the vial to dissolve the protein.

Detailed protocol:

• Centrifuge the vial briefly before opening to ensure all powder is at the bottom.
• Add sterile distilled water (or as specified in the product datasheet) to reach the desired concentration (e.g., for 100 µg protein, add 200–1000 µL for 0.5–0.1 mg/mL).
• Dissolve gently by swirling or inverting; do not vortex or pipette vigorously to avoid denaturation.
• If the protein is to be used at lower concentrations or stored for more than a few hours, add a carrier protein such as 0.1% BSA or HSA to minimize adsorption and stabilize the protein.
• Aliquot the reconstituted protein to avoid repeated freeze-thaw cycles.
• Store aliquots at –20°C to –80°C for long-term storage; for short-term use, 4°C is acceptable for up to a week.
• If the protein was lyophilized from PBS, you may dilute further in cell culture medium or PBS as needed for your assay.

Additional notes:

• Always consult the specific Certificate of Analysis (CoA) or product datasheet for any protein-specific instructions, as buffer composition and optimal reconstitution conditions may vary.
• For functional cell culture assays, ensure the endotoxin level is suitable for your application (typically <0.1 EU/µg for sensitive cell types).
• If using for binding or functional assays, confirm the biological activity post-reconstitution as described in the datasheet.

Summary of best practices:

• Use sterile technique throughout.
• Avoid repeated freeze-thaw cycles.
• Use carrier protein for low-concentration or long-term storage.
• Check datasheet/CoA for any protein-specific requirements.

This protocol ensures optimal solubility and activity of recombinant CTLA-4 for cell culture experiments.