Recombinant Human CD30 Ligand

Recombinant Human CD30 Ligand (CD30L) is a valuable tool in research because it enables precise investigation of CD30/CD30L-mediated signaling, which is central to immune cell activation, proliferation, differentiation, and apoptosis, particularly in T and B lymphocytes. Using the recombinant form allows for controlled, reproducible studies of these pathways in vitro and in vivo, supporting applications in immunology, oncology, and cell therapy research.

Key scientific applications and rationale include:

• Immune Cell Co-stimulation and Activation: CD30L acts as a co-stimulatory molecule for T cells, enhancing their proliferation and cytokine secretion, especially in the context of suboptimal T cell receptor (TCR) stimulation. This is critical for dissecting T cell activation mechanisms and studying Th2-biased immune responses.
• B Cell Function and Humoral Immunity: CD30L can directly stimulate B cell proliferation, differentiation, and antibody production, making it useful for exploring humoral immune responses and secondary antibody production.
• Cancer Research: CD30/CD30L interactions are implicated in the pathophysiology of Hodgkin’s lymphoma, anaplastic large cell lymphoma, and other CD30+ malignancies. Recombinant CD30L can be used to model tumor cell proliferation, apoptosis, and to test therapeutic interventions targeting this pathway.
• Apoptosis and Cell Death Studies: CD30L engagement can induce apoptosis in certain cell types, allowing researchers to study mechanisms of activation-induced cell death and negative selection, particularly in thymic T cells.
• Inflammation and Autoimmunity: CD30L is involved in regulating immune responses in chronic inflammation and autoimmune conditions, making recombinant CD30L a tool for modeling these diseases and testing potential therapies.
• Standardization and Reproducibility: Recombinant proteins provide a consistent, defined reagent for functional assays, ELISA standards, and cell culture experiments, reducing variability compared to primary cell-derived ligands.

Summary of best practices:

• Use recombinant CD30L to stimulate or block CD30 signaling in cell-based assays.
• Employ as a standard in ELISA or cytokine secretion assays.
• Apply in co-culture systems to dissect cell-cell interactions mediated by CD30/CD30L.
• Select carrier-free or BSA-containing formulations based on downstream application requirements.

In summary, recombinant human CD30 ligand is essential for mechanistic studies of immune regulation, cancer biology, and therapeutic development involving the CD30/CD30L axis.

Yes, you can use recombinant Human CD30 Ligand as a standard for quantification or calibration in ELISA assays, provided it is sufficiently purified and its concentration is accurately determined. Recombinant proteins are commonly used as standards in ELISA to generate a standard curve for quantification of the target analyte.

Key considerations for using recombinant Human CD30 Ligand as an ELISA standard:

• Purity and Quantification: The recombinant protein should be highly purified. Accurate quantification of its concentration (e.g., by absorbance at 280 nm, BCA assay, or HPLC) is essential for reliable calibration.
• Standard Curve Preparation: Prepare serial dilutions of the recombinant CD30 Ligand to cover the expected range of concentrations in your samples. Typical standard curves for ELISA range from low picogram to nanogram per milliliter concentrations, depending on assay sensitivity.
• Matrix Effects: Ideally, dilute the recombinant standard in the same buffer or matrix as your samples to minimize matrix effects and ensure comparable binding and detection.
• Validation: Confirm that the recombinant CD30 Ligand behaves similarly to the native protein in your assay system. This includes comparable binding to capture/detection antibodies and similar signal generation.
• Lot-to-Lot Consistency: Use a new standard preparation for each assay run to ensure consistency and accuracy.

Protocol best practices:

• Reconstitute lyophilized recombinant protein according to manufacturer or lab-specific instructions.
• Store aliquots at recommended conditions to avoid repeated freeze-thaw cycles.
• Include replicates of each standard concentration to assess assay precision.

Limitations and caveats:

• If the recombinant protein is not full-length or lacks post-translational modifications present in native samples, quantification may be less accurate.
• Always verify that the recombinant standard is compatible with your specific ELISA antibodies and detection system.

Summary Table:

In conclusion: Recombinant Human CD30 Ligand is suitable as an ELISA standard if properly prepared and validated, enabling quantitative calibration of your assay.

Recombinant Human CD30 Ligand has been validated in published research for several key applications, primarily in immunology, oncology, and cell biology. The most common validated uses include:

• T cell co-stimulation and activation assays: Recombinant CD30 Ligand has been shown to co-stimulate T cells, acting as a mitogen and promoting proliferation, activation, and cytokine secretion (e.g., IL-6, IL-8) in both primary T cells and lymphoma-derived cell lines.
• Functional studies in lymphoma models: It has been used to study cell proliferation, survival, and apoptosis in Hodgkin's disease and other CD30+ lymphoma cell lines, as well as to investigate the CD30/CD30L signaling axis in B-cell non-Hodgkin lymphoma.
• In vitro and in vivo therapeutic targeting: Recombinant CD30 Ligand has been employed to validate therapeutic strategies, such as blocking the CD30/CD30L interaction to impair lymphoma cell viability, and in the development of targeted therapies using peptides or antibodies.
• Cytokine induction assays: Its ability to induce cytokine secretion (e.g., IL-6, IL-8) in human PBMCs and specific cell lines is a standard method for validating its biological activity.
• Apoptosis and cell death studies: CD30L-mediated signaling has been used to induce and study apoptosis in CD30+ cells, including thymic negative selection and lymphoma cell death.
• ELISA and immunoassay standards: Recombinant CD30 Ligand is used as a standard in ELISA and other immunoassays to quantify CD30 or CD30L in biological samples.
• Cell signaling and differentiation studies: It is used to dissect downstream signaling pathways (e.g., NF-κB activation, Th2 differentiation) following CD30 ligation.

Additional validated applications include:

• Flow cytometry (FC): For detection and quantification of CD30L expression or binding in human samples.
• Western blotting and mass cytometry: For protein detection and quantification in research settings.
• Spatial biology and vaccine development research: As a tool for studying tissue localization and immune responses.

Summary Table of Validated Applications

These applications are supported by peer-reviewed studies and product validation data, confirming the recombinant protein's utility in both basic research and translational/therapeutic contexts.

To reconstitute and prepare Recombinant Human CD30 Ligand protein for cell culture experiments, follow these steps to ensure protein integrity and biological activity:

1. Centrifuge the vial briefly (e.g., 3000 rpm for 5 minutes) before opening to collect all lyophilized powder at the bottom and minimize loss.

2. Reconstitution:

   • Add sterile distilled or deionized water to the vial to achieve a concentration of at least 100 μg/mL (0.1 mg/mL) or higher, as recommended for most recombinant cytokines.
   • Gently mix by swirling or inverting; do not vortex or shake vigorously, as this can denature the protein and reduce activity.
   • Allow the solution to sit at room temperature for 15–30 minutes to ensure complete dissolution.

3. Dilution for cell culture:

   • For working solutions, further dilute the reconstituted stock in cell culture medium or a buffer containing a carrier protein (e.g., 0.1% BSA, 10% FBS, 5% HSA, or 5% trehalose) to prevent adsorption and loss of protein activity, especially at low concentrations.
   • Prepare aliquots to avoid repeated freeze-thaw cycles.

4. Storage:

   • After reconstitution, store the stock solution at 2–8°C for up to one week.
   • For longer-term storage, aliquot and freeze at –20°C or –80°C with carrier protein or stabilizer.
   • Avoid repeated freeze-thaw cycles to maintain protein activity.

5. Application:

   • For cell culture assays, typical working concentrations range from 10–100 ng/mL, but optimal concentrations should be determined empirically for your specific experimental setup.
   • Add the protein directly to the culture medium as needed, using freshly thawed aliquots for each experiment.

Key technical notes:

• Do not vortex or use vigorous pipetting at any stage.
• Use carrier proteins for dilution and storage to prevent protein loss due to adsorption.
• Always follow any specific instructions provided in the product’s Certificate of Analysis or technical datasheet for optimal results.

This protocol ensures the recombinant CD30 Ligand remains biologically active and suitable for cell culture experiments.