Recombinant Human CD27

Recombinant Human CD27 is widely used in research applications to study immune cell signaling, costimulatory pathways, and therapeutic targeting due to its critical role as a co-stimulatory receptor in the TNF receptor superfamily. It is particularly valuable for dissecting T cell activation, memory formation, and immune checkpoint regulation.

Key scientific reasons to use recombinant human CD27 include:

• Immune Cell Activation and Costimulation: CD27 is the sole receptor for CD70 and is constitutively expressed on most T cells, memory B cells, plasma cells, and NK cells. The CD27/CD70 interaction is essential for T cell activation, proliferation, survival, and maturation, acting as a costimulatory immune checkpoint alongside TCR engagement.

• Cancer Immunology and Therapeutic Targeting: CD27 is expressed in various hematologic malignancies, and its signaling supports tumor cell proliferation and stemness. Recombinant CD27 is used to screen and characterize monoclonal antibodies targeting the CD27/CD70 axis, which is a promising strategy in cancer immunotherapy, including CAR-T cell engineering and antibody-mediated therapies.

• Functional Assays and Antibody Screening: Recombinant CD27 enables high-throughput screening of anti-CD27 and anti-CD70 antibodies by ELISA and competition assays, facilitating the development of diagnostic and therapeutic reagents.

• Biomarker and Immune Profiling: CD27 serves as a biomarker for immune cell subsets, particularly memory B cells, and is upregulated in various disease states, providing insights into immune heterogeneity and disease progression.

• Mechanistic Studies: Recombinant CD27 is used to investigate the TRAF2-TRAF6-NF-κB signaling pathway, which is triggered by CD27/CD70 interaction and is central to T cell effector function and memory formation.

• Cellular Interaction Research: CD27 and its ligand are important for studying cellular interactions between T and B lymphocytes, as well as the regulation of immune responses.

Typical applications include:

• ELISA and Western blot for antibody screening and quantification.
• Functional assays to study T cell activation, proliferation, and cytokine production.
• Flow cytometry for immune cell subset identification and profiling.
• Preclinical models for evaluating immunotherapeutic strategies targeting the CD27/CD70 axis.

In summary, recombinant human CD27 is a versatile tool for immunology research, cancer biology, antibody development, and therapeutic innovation, enabling precise interrogation of immune costimulatory mechanisms and the development of novel immunotherapies.

You can use recombinant human CD27 as a standard for quantification or calibration in ELISA assays, provided it is appropriately validated and matches the assay’s requirements. Recombinant proteins are commonly used as standards in ELISA because they allow for the generation of a standard curve with known concentrations, enabling accurate quantification of the target protein in your samples.

Key considerations:

• Purity and Formulation: The recombinant CD27 should be highly purified and, ideally, formulated for use as an ELISA standard. Some recombinant CD27 proteins are specifically labeled as suitable for ELISA or sELISA applications.
• Validation: Ensure the recombinant standard is immunologically equivalent to the native protein recognized by the antibodies in your ELISA kit. Some ELISA kits are validated to detect both natural and recombinant human CD27.
• Standard Curve Preparation: Prepare the standard curve using serial dilutions of the recombinant protein, following best practices for accuracy and reproducibility.
• Lot-to-Lot Variability: Be aware that different lots of recombinant protein may show slight differences in immunoreactivity, which can affect quantification. It is recommended to assign the value of your standard based on its performance in the ELISA rather than relying solely on the mass stated on the vial.
• Kit Compatibility: If you are using a commercial ELISA kit, check the kit documentation to confirm that recombinant CD27 is an accepted standard. Many kits are designed to work with both natural and recombinant forms, but this should be verified for your specific assay.

Summary Table: Recombinant CD27 as ELISA Standard

In summary, recombinant human CD27 is suitable as a standard for ELISA quantification if it is validated for your assay and handled according to best practices for standard preparation and calibration.

Research Applications of Recombinant Human CD27

Recombinant human CD27 has been validated for several important applications in published research, spanning immunological screening, therapeutic development, and cellular engineering.

Antibody Screening and Characterization

Soluble CD27-Fc fusion proteins have been extensively used for screening and characterization of monoclonal antibodies. Specifically, these recombinant proteins enable screening of high-affinity anti-human CD27 monoclonal antibodies by ELISA and facilitate competition assays for anti-human CD70 monoclonal antibodies. This application is fundamental for developing therapeutic antibodies targeting the CD27/CD70 axis.

CAR-T Cell Engineering

One of the most significant validated applications involves incorporating CD27 costimulatory signaling into chimeric antigen receptor (CAR)-T cell constructs. Research has demonstrated that human T cells transduced with CAR constructs containing CD27 costimulatory motifs show substantially enhanced antigen-stimulated effector functions in vitro, including increased cytokine secretion and cytotoxicity compared to CAR-T cells with CD3ζ signaling alone. Furthermore, CD27-bearing CAR-T cells demonstrated heightened persistence after infusion in vivo, facilitating improved regression of human cancer in xenogeneic allograft models, with persistence levels comparable to 4-1BB costimulation and superior to CD28 costimulation.

Functional Assays and Binding Studies

Recombinant CD27 proteins have been validated for functional assays measuring inhibition of T cell proliferation induced by CD27 ligand and anti-CD3 stimulation, with established dose-response parameters. These assays provide quantitative measures of CD27-CD70 axis engagement and are essential for evaluating therapeutic candidates.

Biomarker and Diagnostic Applications

CD27 has emerged as a promising biomarker for immune microenvironment characterization, offering insights into new therapeutic approaches for various disease contexts.

To properly reconstitute and prepare Recombinant Human CD27 protein for cell culture experiments, follow these general best practices, which are consistent across major suppliers and technical resources:

1. Reconstitution Protocol

• Centrifuge the vial before opening to ensure all lyophilized powder is at the bottom.
• Reconstitute with sterile, deionized water or the recommended buffer (often specified in the Certificate of Analysis or product manual). For most recombinant CD27 proteins, sterile water or a mild buffer (e.g., PBS, Tris-HCl) is suitable.
• Target concentration: Reconstitute to a stock concentration between 0.1–1.0 mg/mL (e.g., 100 µg protein in 100–1000 µL buffer).
• Gently mix the solution by swirling or pipetting up and down. Avoid vigorous shaking to prevent denaturation.
• If flakes or particulates remain, let the solution mix at room temperature for 15–30 minutes or overnight at 4°C on a rocker.

2. Preparation for Cell Culture

• Aliquot the reconstituted protein into small volumes to avoid repeated freeze-thaw cycles.
• For short-term use (≤1 week): Store aliquots at 2–8°C.
• For long-term storage: Add carrier protein (e.g., 0.1% BSA, 5% HSA, or 10% FBS) and/or 5–50% glycerol (final concentration), then aliquot and store at –20°C to –80°C.
• For serum-free or in vivo experiments: Use trehalose instead of animal-derived carrier proteins.

3. Working Dilution for Cell Culture

• Dilute the stock solution in cell culture medium or appropriate buffer to the desired working concentration (e.g., 10–100 ng/mL, depending on your experimental needs).
• Always use carrier protein (e.g., 0.1% BSA) in the dilution buffer to prevent protein loss due to adsorption to tube walls.

4. General Tips

• Avoid repeated freeze-thaw cycles.
• Use a manual defrost freezer for long-term storage.
• Check the product-specific Certificate of Analysis (CoA) for any unique requirements.

Example Protocol:

1. Centrifuge vial (3000–3500 rpm, 5 min).
2. Add 100 µL sterile water to 100 µg lyophilized CD27 protein (1 mg/mL stock).
3. Gently mix until fully dissolved.
4. Aliquot into small tubes.
5. For immediate use: Store at 2–8°C for up to 1 week.
6. For long-term: Add 0.1% BSA and 10% glycerol, aliquot, and store at –80°C.

Always refer to the product manual or CoA for protein-specific instructions, as some recombinant CD27 proteins may have unique requirements (e.g., tag, expression system, buffer composition).