Recombinant Human B7-H2

Recombinant Human B7-H2 is widely used in research applications to study immune regulation, particularly T cell activation, differentiation, and co-stimulation. Its use is justified by several key scientific advantages:

• Critical Role in T Cell Co-stimulation: B7-H2 (also known as ICOSL or CD275) is a co-stimulatory ligand for both ICOS and CD28 receptors on activated T cells. It is essential for optimal T cell proliferation, cytokine production, and survival, making it a central molecule for dissecting T cell responses in vitro and in vivo.

• Functional Studies: Recombinant B7-H2 can be used to stimulate T cells in the presence of anti-CD3, leading to increased proliferation and cytokine secretion (e.g., IL-4, IFN-γ). This is valuable for:

  • Assessing T cell activation pathways.
  • Investigating mechanisms of immune tolerance or autoimmunity.
  • Evaluating the effects of therapeutic agents targeting co-stimulatory pathways.

• Receptor Binding Assays: Recombinant B7-H2 enables precise analysis of its interactions with ICOS, CD28, and CTLA-4, allowing for mapping of binding domains and quantification of ligand-receptor affinity.

• Cellular Expression and Regulation: B7-H2 is expressed on B cells, dendritic cells, macrophages, endothelial cells, and epithelial cells, and its expression is regulated by cytokines such as TNF-α and IL-1β. Recombinant protein facilitates studies on cell-specific expression and regulation under various conditions.

• Immunotherapy and Disease Models: Because B7-H2 is implicated in T cell-mediated diseases (autoimmunity, cancer, transplantation), recombinant protein is used to model disease mechanisms and to screen for potential immunomodulatory drugs.

• Assay Standardization: Recombinant B7-H2 provides a consistent, well-characterized reagent for ELISA standards, flow cytometry controls, and functional assays, ensuring reproducibility and reliability in experimental results.

Technical Advantages of Recombinant Proteins:

• Sequence Verification and Lot-to-Lot Consistency: Recombinant production allows for precise control over protein sequence and structure, minimizing batch variability and enhancing experimental reproducibility.
• Optimized Functional Activity: Recombinant B7-H2 can be engineered for higher purity, stability, and activity, which is critical for sensitive assays and mechanistic studies.

Typical Applications:

• T cell activation and differentiation assays.
• Cytokine secretion profiling.
• Receptor-ligand binding studies.
• Immunological disease modeling.
• Standardization of immunoassays.

In summary, using Recombinant Human B7-H2 in research provides a robust tool for dissecting immune co-stimulation, modeling disease, and developing immunotherapies, with technical benefits that ensure reproducibility and reliability in experimental outcomes.

Yes, recombinant human B7-H2 can be used as a standard for quantification or calibration in ELISA assays, provided it is of high purity and its concentration is accurately known. This is a common practice in quantitative ELISA, where a standard curve is generated using known concentrations of a purified or recombinant protein to enable precise quantification of the target analyte in samples.

Key considerations and best practices:

• Purity and Characterization: The recombinant B7-H2 protein used as a standard should be highly purified and well-characterized to ensure accurate quantification.
• Concentration Determination: The concentration of the recombinant standard must be determined precisely, typically by absorbance at 280 nm or another validated protein quantification method.
• Standard Curve Preparation: Prepare a dilution series covering the expected range of your samples (e.g., 0–1000 pg/mL or as appropriate for your assay sensitivity).
• Matrix Matching: Ideally, the standard should be diluted in the same buffer or matrix as your samples to minimize matrix effects and ensure parallelism between standard and sample curves.
• Validation: Confirm that the antibodies used in your ELISA recognize both the recombinant standard and the native protein in your samples with similar affinity, ensuring accurate quantification.
• Reconstitution and Handling: If the recombinant protein is supplied lyophilized, follow the manufacturer’s reconstitution instructions carefully, as improper handling can affect protein integrity and quantification accuracy.

Limitations:

• Recombinant proteins may differ from native proteins in post-translational modifications or folding, which can sometimes affect antibody recognition. It is important to validate that your ELISA detects both forms equivalently.
• Recombinant proteins intended as ELISA standards are not always suitable for use in functional bioassays unless specifically validated for such applications.

Summary Table:

In conclusion, recombinant human B7-H2 is suitable as an ELISA standard if these best practices are followed and the protein is validated for this use in your specific assay system.

Recombinant Human B7-H2 has been validated for several key applications in published research, primarily in the context of immunology and cell biology. The most prominent validated applications include:

• T cell costimulation assays: Recombinant B7-H2 is widely used to study its role as a costimulatory ligand for ICOS and CD28, promoting T cell proliferation and cytokine production in vitro. For example, recombinant B7-H2 Fc chimera stimulates human T cell proliferation in the presence of anti-CD3, demonstrating its functional activity in costimulation assays.

• Flow cytometry: Recombinant B7-H2, as well as antibodies targeting B7-H2, are validated for flow cytometric analysis to detect surface expression on various cell types, including monocyte-derived dendritic cells, B cells, and epithelial cells. This application is essential for characterizing immune cell populations and their activation status.

• Blocking/neutralization studies: Recombinant B7-H2 and its antibodies are used in blocking experiments to dissect the functional interactions between B7-H2 and its receptors (ICOS, CD28, CTLA-4). These studies often involve the use of recombinant protein or blocking antibodies to inhibit T cell proliferation or cytokine production, confirming the specificity and biological relevance of B7-H2-mediated signaling.

• ELISA standard: Recombinant B7-H2 is validated as a standard in ELISA assays for quantifying B7-H2 protein levels in biological samples.

• Cell culture experiments: Recombinant B7-H2 is used to modulate immune responses in vitro, such as stimulating T cells or assessing the effects of B7-H2 on dendritic cell function.

• Protein-protein interaction studies: Recombinant B7-H2 is employed in binding assays to characterize its interactions with ICOS, CD28, and CTLA-4, often using fusion proteins or tagged constructs for detection.

Supporting details:

• B7-H2 is a ligand for ICOS, CD28, and CTLA-4, and its recombinant form is used to validate these interactions in human systems, as mouse homologs do not always conserve these binding properties.
• Functional assays using recombinant B7-H2 demonstrate its ability to costimulate T cell proliferation and cytokine secretion, confirming its biological activity.
• Flow cytometry validation includes both direct staining and epitope mapping, with recombinant B7-H2 or anti-B7-H2 antibodies used to assess expression and specificity.
• ELISA validation ensures recombinant B7-H2 can serve as a quantitative standard for protein detection in immunoassays.

Additional relevant information:

• Recombinant B7-H2 is also used in studies of immune regulation, such as maternal–fetal immunity and tumor microenvironment modulation, highlighting its broader relevance in immunological research.
• The protein is available in various formats (Fc chimera, His-tag, biotinylated), allowing flexibility for different assay requirements.

In summary, recombinant human B7-H2 is validated for use in T cell costimulation assays, flow cytometry, blocking/neutralization studies, ELISA standards, cell culture experiments, and protein-protein interaction studies, with extensive published research supporting these applications.

To reconstitute and prepare Recombinant Human B7-H2 protein for cell culture experiments, dissolve the lyophilized protein in sterile water or buffer to the recommended concentration, then dilute with a carrier protein-containing solution (such as 0.1% BSA or 10% FBS in PBS) to stabilize the protein and prevent adsorption or loss.

Detailed protocol:

• Reconstitution:

  • Allow the lyophilized B7-H2 protein vial to equilibrate to room temperature before opening to prevent condensation.
  • Add sterile, distilled water or the buffer specified in the product datasheet (commonly PBS or Tris-buffered saline) to achieve the desired stock concentration (e.g., 0.25 mg/mL is typical for B7-H2).
  • Gently mix by pipetting or slow inversion. Avoid vigorous shaking or vortexing, which can denature the protein or cause foaming.
  • Let the solution stand at room temperature for 15–30 minutes with gentle agitation to ensure complete dissolution.

• Stabilization and Dilution:

  • For cell culture applications, dilute the reconstituted protein in a buffer containing a carrier protein such as 0.1% BSA, 10% FBS, or 5% HSA to minimize adsorption to plastic and stabilize the protein.
  • If using serum-free conditions or for in vivo work, use a non-animal stabilizer such as trehalose instead of BSA or FBS.
  • Prepare working aliquots at the desired concentration for your assay (e.g., 0.3–1.5 μg/mL for functional T cell co-stimulation assays).

• Storage:

  • Aliquot the reconstituted protein to avoid repeated freeze-thaw cycles, which can degrade the protein.
  • Store aliquots at −20°C to −80°C for long-term storage, or at 2–8°C for short-term use (up to one week).
  • For long-term storage, adding 5–50% glycerol can further stabilize the protein.

Key technical notes:

• Always use sterile technique to avoid contamination.
• Avoid repeated freeze-thaw cycles by aliquoting.
• For functional cell culture assays, confirm the optimal working concentration for your specific application, as this can vary depending on cell type and assay design.
• If the protein is tagged (e.g., His-tag, Fc-tag), ensure compatibility with your downstream application.

Summary Table:

This protocol ensures the stability and bioactivity of recombinant B7-H2 for cell culture experiments. Always consult the specific product datasheet for any unique recommendations.