Recombinant Human PD-L1 (B7-H1)

Recombinant Human PD-L1 (B7-H1) is a valuable tool for a wide range of research applications, particularly in immunology and cancer biology. Here are several key reasons why you should consider using Recombinant Human PD-L1 in your research:

1. Study of Immune Checkpoint Pathways

PD-L1 (B7-H1) is a critical immune checkpoint molecule that interacts with PD-1 on T cells to regulate immune responses. Using recombinant PD-L1 allows you to:

• Investigate the molecular mechanisms of PD-1/PD-L1 interactions.
• Study how this pathway contributes to immune tolerance, T cell anergy, and immune evasion by tumors.

2. Functional Assays and Bioassays

Recombinant PD-L1 is suitable for various functional assays, including:

• Binding assays: To measure the affinity and specificity of antibodies, peptides, or small molecules targeting PD-L1.
• Cell proliferation and activation assays: To assess the impact of PD-L1 on T cell function and proliferation.
• Enzymatic activity studies: To explore downstream signaling events triggered by PD-L1 engagement.

3. Development and Testing of Therapeutics

Recombinant PD-L1 is essential for:

• Screening and characterizing potential inhibitors, antibodies, or peptides that block the PD-1/PD-L1 interaction.
• Evaluating the efficacy of novel immunotherapeutic agents designed to disrupt this pathway.

4. Immune Tolerance and Autoimmunity Research

PD-L1 plays a crucial role in maintaining immune tolerance and preventing autoimmunity. Using recombinant PD-L1 enables you to:

• Study the development of regulatory T cells (Tregs) and their suppressive functions.
• Investigate the balance between immune activation and suppression in different disease contexts.

5. Cancer Immunotherapy

PD-L1 is highly expressed in many human cancers and is a key target for cancer immunotherapy. Recombinant PD-L1 can be used to:

• Model the tumor microenvironment and study how PD-L1 expression affects tumor growth and immune evasion.
• Develop and test strategies to enhance antitumor immunity by blocking PD-L1.

6. High Purity and Bioactivity

Recombinant Human PD-L1 is typically produced with high purity and guaranteed bioactivity, ensuring reliable and reproducible results in your experiments. This is crucial for:

• Consistent performance across different batches.
• Accurate quantification and comparison of experimental outcomes.

7. Versatility in Applications

Recombinant PD-L1 can be used in a variety of applications, including:

• ELISA and other immunoassays.
• Flow cytometry and cell surface staining.
• Protein-protein interaction studies.
• Structural and biochemical analyses.

8. Support for Preclinical and Clinical Research

Recombinant PD-L1 is widely used in preclinical studies to validate therapeutic targets and in clinical research to develop biomarkers for predicting response to immunotherapy.

In summary, Recombinant Human PD-L1 (B7-H1) is an indispensable reagent for researchers studying immune checkpoint pathways, developing new immunotherapies, and exploring the complex interplay between the immune system and cancer. Its high purity, bioactivity, and versatility make it a reliable choice for a broad range of experimental applications.

Yes, recombinant human PD-L1 (B7-H1) can be used as a standard for quantification or calibration in ELISA assays, provided it is of high purity, properly quantified, and compatible with your assay’s antibody pair and detection system.

Key considerations and supporting details:

• Recombinant PD-L1 is commonly used as a standard in ELISA kits designed to quantify PD-L1 in biological samples. Many commercial ELISA kits for human PD-L1 use HEK293-expressed recombinant human PD-L1/B7-H1 as the calibrator or standard, and these kits demonstrate that the recombinant protein yields standard curves that are parallel to those generated with natural PD-L1 from biological samples.

• Purity and quantification: The recombinant PD-L1 should be highly purified (typically >90–95% by SDS-PAGE) and accurately quantified, as impurities or inaccurate concentration will affect the reliability of your standard curve.

• Expression system: Most validated standards are expressed in mammalian systems (e.g., HEK293 cells) to ensure proper folding and post-translational modifications, which can affect antibody recognition in ELISA.

• Assay compatibility: The recombinant PD-L1 must be recognized by both the capture and detection antibodies used in your ELISA. Most sandwich ELISAs for PD-L1 are validated to detect both natural and recombinant forms, but it is essential to confirm this for your specific antibody pair.

• Standard curve preparation: Prepare a dilution series of the recombinant PD-L1 in the same buffer or matrix as your samples to generate a standard curve for quantification.

• Documentation: If using a recombinant PD-L1 not supplied with your ELISA kit, ensure you have documentation on its concentration, purity, and source, and ideally validate its performance in your assay by comparing standard curves with those from a commercial kit, if available.

Limitations and best practices:

• If your recombinant PD-L1 is not from the same source or expression system as the kit standard, minor differences in glycosylation or folding could affect antibody binding and quantification accuracy. Parallelism testing (comparing standard curves from recombinant and natural PD-L1) is recommended to confirm equivalence.

• Some suppliers note that their recombinant proteins are suitable for ELISA standard curves but not for functional bioassays unless specifically validated for that purpose.

In summary, recombinant human PD-L1 is widely used and accepted as a standard for ELISA quantification, provided it is properly validated and compatible with your assay system.

Recombinant Human PD-L1/B7-H1 proteins have been validated for a diverse range of applications in published research, reflecting their importance in immunological and cancer biology studies.

Primary Research Applications

Binding and Protein-Protein Interaction Studies

Recombinant PD-L1 proteins are extensively used in binding assays to characterize interactions with their natural ligands and engineered binding partners. These applications include screening high-affinity monoclonal antibodies against PD-L1 by ELISA and competition-based assays to identify anti-PD-1 antibodies. Biotinylated variants have been specifically optimized for biopanning experiments and other binding validation studies.

Bioassay and Functional Assessment

Functional bioassays represent a major application category, where recombinant PD-L1 is used to evaluate immunomodulatory effects. Notably, the bioactivity of PD-L1 Fc chimera proteins has been validated through their ability to inhibit anti-CD3 antibody-induced IL-2 secretion in human T lymphocyte primary cells, with reported effective concentrations (ED50) in the range of 0.075-0.75 μg/mL.

Cell-Based Applications

Recombinant PD-L1 has been validated for applications involving stem cell and immune cell maintenance or differentiation, as well as cell adhesion studies. Cell proliferation assays represent another validated application, where PD-L1's immunoregulatory effects on T cell proliferation can be measured.

Immunological Detection and Analysis

Surface plasmon resonance has been employed to characterize PD-L1 interactions in real-time kinetic studies. Additionally, recombinant PD-L1 serves as a control reagent for SDS-PAGE analysis and protein characterization.

Therapeutic Development Context

Beyond basic research, recombinant PD-L1 proteins have supported the development of bifunctional fusion proteins targeting both PD-L1 and transforming growth factor-beta for anticancer immunotherapy, and have been instrumental in characterizing bispecific antibodies that simultaneously engage PD-L1 and other immune checkpoint pathways.

To reconstitute and prepare Recombinant Human PD-L1 (B7-H1) protein for cell culture experiments, dissolve the lyophilized protein in sterile PBS at a concentration of 100 μg/mL for Fc-chimera formats, or as specified by the manufacturer for other formats. For His-tagged versions, reconstitution at 500 μg/mL in PBS is recommended. Always use sterile technique and avoid repeated freeze-thaw cycles.

Step-by-step protocol:

• Centrifuge the vial briefly (e.g., 3000 rpm for 5 min) before opening to ensure all powder is at the bottom.
• Add sterile PBS (or sterile water if specified for your format) to achieve the desired concentration (commonly 100 μg/mL for Fc-chimera, 500 μg/mL for His-tag).
• Gently mix by pipetting up and down or by slow vortexing. Avoid vigorous agitation to prevent protein denaturation.
• Allow the solution to sit at room temperature for 10–15 min to ensure complete dissolution.
• Aliquot the solution into sterile tubes to minimize freeze-thaw cycles.
• Storage:
  • Short-term (≤1 week): 2–8 °C under sterile conditions.
  • Long-term (≤3 months): –20 °C to –70 °C under sterile conditions.
  • Avoid repeated freeze-thaw cycles; use a manual defrost freezer.

Additional notes for cell culture experiments:

• Confirm the endotoxin level is suitable for cell culture (typically <0.1 EU/μg for sensitive immune assays).
• If required, further dilute the reconstituted stock in cell culture medium immediately before use.
• For functional assays, typical working concentrations range from 0.01–10 μg/mL, but optimize based on your experimental design.

Summary Table:

Always refer to the specific product datasheet for any format-specific instructions. If the datasheet recommends sterile water instead of PBS, use sterile water. For cell culture, ensure all solutions are sterile and endotoxin-free.