Recombinant Human CD137L (4-1BBL)

Recombinant Human CD137L (4-1BBL) is used in research applications primarily to study and manipulate immune cell activation, proliferation, and survival, especially in the context of T cell and NK cell biology, immunotherapy, and vaccine development.

Key scientific reasons to use recombinant human CD137L (4-1BBL) include:

• Potent T Cell Costimulation: CD137L is a critical costimulatory ligand for CD137 (4-1BB), a receptor expressed on activated T cells. The CD137/CD137L interaction triggers NF-κB-dependent signaling, which robustly augments T cell activation, proliferation, and survival. This is essential for generating strong and sustained immune responses.

• Enhancement of Memory T Cell Formation: Engagement of 4-1BBL not only supports effector T cell responses but also promotes the generation and maintenance of memory T cells, which are crucial for long-term immunity.

• Immunotherapy Research: Recombinant CD137L has shown promise in preclinical and clinical studies as an immunomodulator in cancer immunotherapy. It can enhance antitumor CD8+ T cell responses, sustain T cell survival, and improve the efficacy of therapeutic vaccines by increasing antigen uptake and cross-presentation by dendritic cells.

• Vaccine Adjuvant and Antigen Delivery: CD137L can be conjugated to antigens to improve their delivery to dendritic cells, leading to more effective antigen presentation and stronger T cell responses. This approach has demonstrated increased efficacy in therapeutic cancer vaccine models.

• Expansion of NK Cells: Immobilized recombinant CD137L, in combination with cytokines like IL-21, can be used to expand natural killer (NK) cells from peripheral blood mononuclear cells, which is valuable for adoptive cell therapy research.

• Screening and Functional Assays: Recombinant CD137L is used in ELISA, flow cytometry, and cell-based assays to screen for antibodies or compounds that modulate the CD137/CD137L pathway, and to validate the functional activity of immune cells or engineered receptors.

• Study of Immune Regulation: The CD137/CD137L axis is involved in modulating regulatory T cell (Treg) function and can reverse T cell anergy, making it a useful tool for dissecting mechanisms of immune tolerance and autoimmunity.

Summary of applications:

• Costimulation and activation of T cells and NK cells
• Enhancement of vaccine efficacy and antigen presentation
• Cancer immunotherapy research
• Functional screening of antibodies and immune modulators
• Studies of immune memory, tolerance, and regulation

Using recombinant human CD137L (4-1BBL) provides a controlled, reproducible means to engage this critical immune pathway in vitro and in vivo, facilitating mechanistic studies and translational research in immunology and oncology.

You can use recombinant human CD137L (4-1BBL) as a standard for quantification or calibration in ELISA assays, provided that the ELISA is specifically designed to detect CD137L and the recombinant protein matches the form of the analyte the assay is intended to quantify.

Key considerations:

• Assay specificity: The ELISA must be validated for the detection of CD137L (4-1BBL), not CD137 (4-1BB) itself. ELISA kits for human 4-1BBL typically use the recombinant extracellular domain of human 4-1BBL as the standard. Using a recombinant human CD137L that matches this domain is appropriate for calibration.
• Protein form: The recombinant protein should be in the same form (e.g., soluble extracellular domain) as the standard used in validated ELISA kits. Most commercial kits use the extracellular domain as the standard.
• Validation: The recombinant CD137L should be well-characterized (purity, identity, and activity confirmed) and ideally validated for use in ELISA, as is common for commercially available recombinant proteins.
• Concentration range: Ensure the concentration range of your standard curve matches the sensitivity and dynamic range of your ELISA (e.g., 0.2–50 ng/mL or as specified by your kit).
• Matrix effects: If you are quantifying CD137L in complex biological samples (e.g., serum, plasma), matrix effects may influence quantification. It is best practice to prepare the standard curve in the same matrix as your samples or to validate recovery in your sample type.

Supporting details:

• Commercial ELISA kits for human 4-1BBL (CD137L) use recombinant extracellular domain protein as the standard for quantification.
• Recombinant human CD137L is validated for use in ELISA and receptor binding assays, and is commonly used as a standard in published protocols.
• The specificity of the assay is critical: using a CD137L standard in a CD137 (receptor) ELISA is not appropriate, and vice versa.

Summary Table: Use of Recombinant Human CD137L as ELISA Standard

If your recombinant CD137L matches these criteria, it is suitable for use as a standard in ELISA quantification or calibration for CD137L (4-1BBL).

Recombinant Human CD137L (4-1BBL) has been validated for a range of applications in published research, primarily in immunological and cancer studies. The most commonly validated applications include:

• Functional Assays: Used to assess the costimulatory activity of CD137L on T cells, including proliferation, cytokine production, and survival enhancement.
• ELISA (Enzyme-Linked Immunosorbent Assay): Utilized for quantifying CD137L or for screening monoclonal antibodies against CD137L or its receptor.
• Western Blot: Applied to detect CD137L protein expression in cell lysates or recombinant preparations.
• Blocking Assays: Used to study the inhibition of CD137/CD137L interactions, often to dissect signaling pathways or immune responses.
• Immunohistochemistry: Employed to localize CD137L expression in tissue samples.
• Flow Cytometry: Validated for detecting CD137L on cell surfaces or for functional readouts using engineered reporter cell lines.
• Cellular Signaling Assays: Used to confirm the ability of recombinant CD137L to trigger downstream signaling (e.g., NF-κB activation) in engineered cell lines.
• Cancer Immunotherapy Models: Recombinant or engineered forms (e.g., SA-4–1BBL) have been used in vivo to stimulate anti-tumor immune responses, prevent tumor development, and control tumor recurrence in mouse models.
• T Cell Costimulation Studies: Immobilized or multimerized forms of recombinant CD137L have been shown to provide potent costimulatory signals to purified T cells, especially when cross-linked or presented in a multivalent format.

Key details and context:

• Functional validation includes demonstration of T cell activation, proliferation, cytokine induction (such as IL-2), and prevention of activation-induced cell death.
• ELISA and flow cytometry are used for both detection and screening of antibodies or ligands interacting with CD137L.
• Blocking assays help dissect the role of CD137L in immune signaling by preventing its interaction with CD137.
• Cancer immunotherapy research has used recombinant CD137L to enhance anti-tumor immunity, both as a monotherapy and as an adjuvant in vaccine models.
• Structural and mechanistic studies have shown that multimerization or immobilization of recombinant CD137L is often required for robust costimulatory activity on T cells.

These applications are supported by both commercial validation and peer-reviewed research, reflecting the broad utility of recombinant human CD137L in immunological, biochemical, and translational studies.

To reconstitute and prepare Recombinant Human CD137L (4-1BBL) protein for cell culture experiments, follow these best-practice steps based on current protocols:

• Centrifuge the vial briefly before opening to ensure all lyophilized material is at the bottom.
• Reconstitute the protein in sterile, distilled water or sterile PBS. The recommended concentration is typically 0.1–1.0 mg/mL.
  • For some formulations, specifically use sterile PBS containing at least 0.1% BSA (bovine or human serum albumin) to enhance stability and prevent adsorption to surfaces.
  • If the product datasheet specifies, use ultrapure water (18 MΩ·cm) for initial reconstitution, then dilute further as needed.
• Do not vortex the solution; gently swirl or invert to dissolve the protein completely.
• Aliquot the reconstituted protein to avoid repeated freeze-thaw cycles, which can degrade protein activity.
• Storage after reconstitution:
  • Short-term (up to 1 week): Store at 2–8°C.
  • Long-term: Dilute in buffer with a carrier protein (e.g., 0.1% BSA), aliquot, and store at –80°C to –20°C.
• Working solution: For cell culture, further dilute the stock solution in your assay buffer or cell culture medium immediately before use. Ensure the final buffer is compatible with your cells (e.g., serum-free or serum-containing medium as appropriate).

Additional notes:

• The optimal working concentration for functional assays should be determined empirically, but typical starting ranges are 0.1–10 μg/mL depending on the application and cell type.
• If using a fusion protein (e.g., Fc or AviTag), confirm the reconstitution buffer is compatible with the fusion partner and downstream applications.
• Always consult the specific product datasheet for any unique instructions or formulation details, as excipients (e.g., trehalose, mannitol, PEG) may affect solubility or storage.

Summary protocol example:

1. Briefly centrifuge the vial.
2. Add sterile PBS (with 0.1% BSA) to achieve 0.1–1.0 mg/mL.
3. Gently mix until fully dissolved.
4. Aliquot and store at –80°C for long-term use.
5. Thaw aliquots as needed, dilute to working concentration in cell culture medium, and use immediately.

These steps will ensure protein stability and activity for cell culture experiments.