Recombinant Human CD40 Ligand (aa 108-261)

Recombinant Human CD40 Ligand (aa 108-261) is widely used in research applications because it is a potent and specific activator of the CD40 receptor, which plays a central role in immune regulation, B cell activation, antibody production, and cellular differentiation.

Key scientific reasons to use this reagent include:

• B Cell Activation and Expansion: CD40 Ligand (CD40L) stimulates proliferation and differentiation of B cells, enabling robust antibody production and immunoglobulin class switching. This is essential for studies on humoral immunity, vaccine responses, and antibody engineering.
• Antigen Presentation Enhancement: CD40L upregulates co-stimulatory molecules on B cells and dendritic cells, improving their capacity to present antigens and activate T cells. This is critical for immunotherapy research and studies involving antigen-specific immune responses.
• Functional Assays and Cell Culture: The aa 108-261 fragment represents the extracellular, receptor-binding domain of CD40L, ensuring high bioactivity and compatibility with in vitro assays, cell culture, and differentiation protocols.
• Cancer and Autoimmunity Research: Recombinant CD40L has demonstrated direct antitumor effects by inducing apoptosis in CD40-expressing carcinoma cells and can be used to study immune modulation in cancer and autoimmune disease models.
• Reduced Immunogenicity and Off-target Effects: Using recombinant human CD40L avoids the depletion of normal immune cells and the risk of anti-mouse antibody responses associated with monoclonal antibody therapies, making it preferable for translational and clinical research.

Typical applications include:

• Cell culture stimulation of B cells, dendritic cells, and monocytes.
• Functional bioassays for immune cell activation and differentiation.
• Studies of antibody production, class switching, and germinal center formation.
• Cancer immunotherapy research, including expansion of antigen-presenting cells.
• Investigation of autoimmune and inflammatory disease mechanisms.

Best practices:
Use the recombinant protein at experimentally determined concentrations for optimal activation (often in the low ng/mL range for binding and proliferation assays). Combine with cytokines (e.g., IL-4) for synergistic effects on B cell differentiation and antibody secretion. Confirm bioactivity using appropriate controls and functional readouts (e.g., proliferation, surface marker expression, cytokine production).

In summary, Recombinant Human CD40 Ligand (aa 108-261) is a versatile and reliable tool for dissecting immune mechanisms, optimizing cell-based assays, and advancing translational research in immunology, oncology, and beyond.

You can use recombinant human CD40 Ligand (aa 108-261) as a standard for quantification or calibration in ELISA assays, provided that the ELISA is designed to recognize this fragment and the standard is well-characterized and quantified.

Key considerations and supporting details:

• Fragment Compatibility: The aa 108-261 region corresponds to the extracellular, soluble portion of CD40 Ligand, which is commonly used in ELISA standards and is recognized by most commercial CD40L ELISA kits. Many kits and published protocols use recombinant CD40L of this length as a standard, indicating its suitability for quantification.

• Assay Design: Ensure that your ELISA antibodies (capture and detection) recognize epitopes within the aa 108-261 region. Most commercial sandwich ELISAs for CD40L are designed to detect both natural and recombinant forms, including this fragment. If using a custom or in-house ELISA, confirm antibody specificity to this region.

• Standard Preparation: The recombinant protein should be highly purified, quantified accurately (e.g., by absorbance at 280 nm or BCA assay), and reconstituted according to manufacturer or protocol instructions. Prepare a standard curve covering the expected concentration range of your samples.

• Validation: It is good practice to validate the standard by running parallel curves with both the recombinant standard and a known positive control (e.g., native CD40L from plasma or cell culture supernatant) to confirm parallelism and accurate quantification.

• Documentation: The recombinant CD40L (aa 108-261) is widely used as a standard in functional and binding ELISAs, with documented binding activity and purity. This supports its use for quantitative calibration.

Summary Table: Use of Recombinant Human CD40L (aa 108-261) as ELISA Standard

Conclusion:
Recombinant human CD40 Ligand (aa 108-261) is suitable as a standard for ELISA quantification if your assay is designed to detect this region and the protein is properly prepared and validated. Always confirm compatibility with your specific ELISA system.

Recombinant Human CD40 Ligand (aa 108-261) has been validated for a broad range of scientific applications in published research, primarily in immunology, cell biology, and translational medicine.

Key validated applications include:

• Cell stimulation and functional bioassays: Used to activate B cells, dendritic cells, and other antigen-presenting cells in vitro, often to study immune cell activation, proliferation, and cytokine production.
• Binding assays and protein-protein interaction studies: Employed in ELISA and biolayer interferometry (BLI) to confirm specific binding to CD40 and characterize ligand-receptor interactions.
• Cell culture: Utilized to maintain or differentiate stem and immune cells, especially for studies of B cell maturation and T cell-dependent responses.
• Ex vivo activation of dendritic cells: Applied to generate antigen-presenting cells for immunotherapy and vaccine development, including cancer immunology research.
• Western blotting: Used as a positive control or to confirm protein expression and purity.
• Mass cytometry (CyTOF) and spatial biology: Incorporated in advanced single-cell and tissue-level analyses to study immune cell phenotypes and interactions.
• Preclinical and clinical research: Investigated as an immunomodulatory agent in cancer therapy, autoimmune disease models, and vaccine studies.

Representative published research applications:

• B cell activation and germinal center formation: Used to stimulate B cells in studies of aging and immune response.
• Regulation of B cell functions in disease models: Applied in experimental autoimmune encephalomyelitis (EAE) to assess immune modulation.
• Proinflammatory cytokine induction: Used to study Th17 cell generation and cytokine production in rheumatoid arthritis models.
• Cancer immunotherapy: Validated in phase I clinical trials for safety, pharmacokinetics, and antitumor activity in cancer patients.
• Cardiovascular and thrombosis research: Utilized to investigate the role of CD40L in vascular inflammation and platelet function.

Summary Table of Validated Applications

Additional context:

• The aa 108-261 fragment corresponds to the soluble, bioactive region of CD40L, commonly used for in vitro and ex vivo studies due to its ability to mimic physiological CD40-CD40L interactions.
• This recombinant protein is frequently used in combination with cytokines (e.g., IL-4) to drive specific immune responses in cell-based assays.
• Its role in immune modulation makes it a valuable tool for both basic research and translational applications, including the development of immunotherapies and vaccines.

If you require detailed protocols or specific assay conditions for any of these applications, please specify the intended use.

To reconstitute and prepare Recombinant Human CD40 Ligand (aa 108-261) for cell culture experiments, dissolve the lyophilized protein in sterile phosphate-buffered saline (PBS) to a concentration of 0.2–0.5 mg/mL, optionally including 0.1% human or bovine serum albumin (BSA) to stabilize the protein.

Step-by-step protocol:

• Centrifuge the vial briefly before opening to ensure all lyophilized material is at the bottom.
• Add sterile PBS (pH 7.2–7.4) directly to the vial to achieve the desired concentration (commonly 0.2–0.5 mg/mL).
• For enhanced stability and to prevent adsorption, include at least 0.1% BSA in the PBS if your application allows.
• Gently mix by pipetting; do not vortex, as vigorous agitation may denature the protein.
• Allow the solution to sit at room temperature for several minutes to ensure complete dissolution.
• Aliquot the reconstituted protein to avoid repeated freeze-thaw cycles, which can reduce activity.
• Storage after reconstitution:
  • Store at 2–8°C for up to 1 month under sterile conditions.
  • For longer-term storage, freeze aliquots at –20°C to –70°C for up to 3 months.
• For cell culture experiments, dilute the stock solution to the working concentration in your assay buffer or culture medium immediately before use. Typical working concentrations range from 0.5–10 μg/mL, depending on cell type and experimental requirements.

Additional notes:

• Avoid repeated freeze-thaw cycles by preparing single-use aliquots.
• If using a formulation with carrier protein (BSA), ensure compatibility with your downstream applications.
• The protein is typically supplied lyophilized from a buffer containing Tris-HCl or NaH₂PO₄, NaCl, and EDTA; check the certificate of analysis for exact formulation.
• For functional assays, the effective dose (ED50) for CD40L activity is typically in the range of 1–3 μg/mL in the presence of 20 ng/mL rhIL-4.

This protocol ensures optimal solubility, stability, and biological activity of recombinant CD40 Ligand for cell culture experiments.