Recombinant Human CD30

Recombinant Human CD30 is widely used in research applications to study immune regulation, cancer biology, and therapeutic development, particularly in the context of lymphoid malignancies and T-cell function. Its use enables precise, reproducible experiments involving CD30-mediated signaling, antibody development, and cellular assays.

Key scientific reasons to use recombinant human CD30 include:

• Modeling CD30-mediated signaling: CD30 is a member of the TNF receptor superfamily and plays a critical role in regulating immune cell proliferation, differentiation, and apoptosis. Recombinant CD30 allows researchers to dissect these pathways in vitro, facilitating mechanistic studies of T-cell and B-cell responses.

• Cancer immunotherapy research: CD30 is highly expressed on certain lymphomas, such as Hodgkin lymphoma and anaplastic large cell lymphoma. Recombinant CD30 is essential for developing and validating CD30-targeted therapies, including monoclonal antibodies and CAR-T cells. It serves as a target antigen in cytotoxicity assays, antibody screening, and CAR-T cell functional studies.

• Antibody and ligand binding assays: Recombinant CD30 provides a standardized antigen for screening and characterizing anti-CD30 antibodies, including therapeutic candidates and diagnostic reagents. It is also used to study interactions with CD30 ligand (CD30L), which mediates diverse effects such as cell proliferation, activation, and apoptosis.

• Cellular activation and differentiation studies: CD30 ligation influences B-cell proliferation, antibody production, and chemokine expression, as well as T-cell differentiation and immune regulation. Recombinant CD30 enables controlled stimulation or inhibition of these processes in cell-based assays.

• High purity and reproducibility: Recombinant proteins are produced under defined conditions, ensuring batch-to-batch consistency, low endotoxin levels, and suitability for sensitive applications such as flow cytometry, ELISA, and cell culture.

Typical applications include:

• ELISA and Western blotting for quantifying CD30 or anti-CD30 antibodies.
• Flow cytometry for detecting CD30 expression or antibody binding.
• Cell-based assays to study CD30 signaling, proliferation, and apoptosis.
• Functional assays for CAR-T cell cytotoxicity and cytokine release.

In summary, recombinant human CD30 is a critical reagent for immunology, oncology, and therapeutic research, enabling precise investigation of CD30 biology and the development of targeted interventions.

Yes, recombinant human CD30 can be used as a standard for quantification and calibration in ELISA assays, with important considerations regarding formulation and application context.

Suitability as an ELISA Standard

Recombinant CD30 proteins are specifically designed and provided as standards in sandwich ELISA kits for measuring both natural and recombinant CD30. The recombinant protein consists of the extracellular domain (amino acids 19-379) and is suitable for establishing standard curves to quantify CD30 in various sample types including serum, plasma, cell culture supernatants, and other biological fluids.

Formulation Considerations

The choice between carrier-free and carrier-protein formulations is critical for your application:

Carrier-protein formulation (with BSA): This version is recommended for use as an ELISA standard. The bovine serum albumin carrier enhances protein stability, increases shelf-life, and allows storage at more dilute concentrations, making it practical for standard curve preparation.

Carrier-free formulation: This format is available when the presence of BSA could interfere with your specific assay requirements. However, carrier-free recombinant proteins are generally not recommended for bioassay applications as they are not typically tested for such uses.

Standard Curve Preparation

When preparing your standard curve, follow these guidelines:

• Use the recombinant protein according to manufacturer reconstitution instructions, as these may be lot-specific
• Standard curves typically range from 0 to 1000 pg/mL, though ranges up to 3000 pg/mL are possible for extremely high target concentrations
• Ensure you use recommended microplates, buffers, diluents, and substrates as specified in the assay protocol to achieve optimal results

Important Limitation

Be aware that recombinant CD30 proteins provided as ELISA standards are not validated for bioassay applications. If your research requires functional assessment of CD30 activity (such as receptor binding or signaling studies), you should use recombinant proteins specifically tested and validated for bioassay purposes rather than those designated solely for ELISA calibration.

Recombinant Human CD30 has been validated for several key applications in published research, primarily in immunology, oncology, and cell biology.

Validated Applications:

• Functional Assays: Recombinant CD30 is used to study its role as a receptor for CD30 ligand (CD30L/TNFSF8), which regulates cellular growth, transformation of activated lymphoblasts, and gene expression via NF-κB activation. It is biologically active and can be used to assess downstream signaling events, such as cytokine secretion and cell proliferation.

• ELISA Standard: Recombinant CD30 is commonly used as a standard in ELISA assays to quantify soluble CD30 in biological samples, aiding in the study of immune activation and lymphoma diagnostics.

• Cell Culture Studies: The protein is used in cell culture to investigate CD30-mediated signaling, including co-stimulation of T cells, induction of cytokine secretion (e.g., IL-13, IL-6), and effects on B cell proliferation and antibody production.

• Immunotherapy Research: Recombinant CD30 is employed in the development and validation of CD30-targeted therapies, such as CAR-T cells and bispecific antibodies for Hodgkin lymphoma and anaplastic large cell lymphoma. It serves as a target antigen for evaluating the specificity and efficacy of engineered immune cells and antibodies.

• Flow Cytometry and Immunoassays: Recombinant CD30 is used as a positive control or antigen in flow cytometry, immunohistochemistry (IHC), immunoprecipitation (IP), and Western blot (WB) assays to detect CD30 expression and study its distribution in various cell types.

• Protein Interaction Studies: It is utilized to characterize interactions with CD30 ligand and downstream signaling molecules, including TRAF1, which augments NF-κB activation.

Additional Notes:

• Biological Activity: Recombinant CD30 is confirmed to be biologically active, with validated activity in functional assays such as stimulation of cytokine secretion and cell proliferation.
• Expression System: Most recombinant CD30 proteins are expressed in HEK293 cells, ensuring proper folding and post-translational modifications relevant for functional studies.
• Disease Models: Recombinant CD30 is used in preclinical models to study its role in lymphoma, autoimmunity, and chronic inflammation.

These applications are supported by multiple peer-reviewed studies and product validation data, confirming the utility of recombinant human CD30 in both basic research and translational immunotherapy development.

Reconstitution Protocol

Recombinant human CD30 protein is typically supplied in lyophilized form and requires proper reconstitution before use in cell culture experiments. The reconstitution process should follow the specific protocol provided in the Certificate of Analysis (CoA) for your particular preparation, as formulations may vary slightly between sources.

Initial Preparation Steps:

Begin by centrifuging the reagent tube before opening the cap. This prevents the lyophilized powder from drifting and adhering to the tube wall or cap during transportation. Allow 15-30 minutes for reconstitution at room temperature with gentle agitation. Avoid vigorous shaking or vortexing, as this can cause foaming and potentially denature the protein.

Reconstitution Concentration:

Reconstitute the protein to a concentration between 0.1-1.0 mg/mL in sterile distilled water or PBS, depending on your experimental requirements. For example, if you have 100 µg of protein, calculate the appropriate volume of reconstitution buffer accordingly. Some preparations recommend reconstituting at 100 µg/mL in PBS, while others suggest 0.1-0.5 mg/mL in sterile distilled water.

Storage and Stability

Short-term Storage:

After reconstitution, store the protein at 2-8°C in a refrigerator under sterile conditions. Under these conditions, the protein remains active for up to one week, which is sufficient for typical 5-7 day experimental cycles.

Long-term Storage:

For extended storage, prepare aliquots and store at -20°C to -70°C in a manual defrost freezer. Avoid repeated freeze-thaw cycles, as these can compromise protein integrity and biological activity. Long-term stability at -70°C is typically maintained for 12 months from the date of receipt.

Preparation for Cell Culture Use

Dilution Strategy:

If you need to work at lower protein concentrations, dilute the reconstituted protein using a carrier protein-containing solution such as 0.1-1% BSA, 10% FBS, or 5% HSA. This prevents the protein from adhering to tube walls and maintains biological activity at dilute concentrations.

Serum-free Applications:

For serum-free culture or in vivo experiments where animal-derived proteins are inappropriate, use trehalose as a carrier protein instead. Trehalose serves as an effective stabilizer for bioactive proteins and can be used for long-term storage at -20°C to -80°C with 5-50% glycerol (final concentration).

Quality Considerations:

Ensure the reconstituted protein maintains >95% purity by SDS-PAGE and has an endotoxin level <0.1 EU/µg, which is typical for recombinant CD30 preparations. The protein is biologically active and suitable for functional assays and cell culture applications.