Recombinant Human/Cynomolgus/Rhesus Macaque CD28

Recombinant Human/Cynomolgus/Rhesus Macaque CD28 is used in research applications to study and manipulate T cell activation, immune co-stimulation, and cross-species immunological interactions, particularly in translational and preclinical models involving non-human primates.

Key reasons to use this recombinant protein include:

• Critical Role in T Cell Activation: CD28 is a primary co-stimulatory receptor on T cells, essential for full T cell activation, proliferation, and survival upon binding to its ligands B7-1 (CD80) and B7-2 (CD86). Without CD28-mediated co-stimulation, T cells fail to mount effective immune responses.

• Cross-Species Compatibility: The recombinant protein is engineered to be identical or highly homologous across human, cynomolgus, and rhesus macaque species, enabling direct comparison and translation of findings between human and non-human primate models. This is particularly important for preclinical studies, vaccine development, and immunotherapy research where non-human primates serve as the most relevant models for human immune responses.

• Applications in Immunological Assays: Recombinant CD28 is widely used in:

  • In vitro T cell activation assays to mimic physiological co-stimulation.
  • Antibody screening and binding studies (e.g., ELISA, SPR, BLI) to characterize interactions with anti-CD28 antibodies or B7 ligands.
  • Cell-based assays for evaluating immune checkpoint modulation, CAR-T cell function, and immunogenicity.
  • Protein-protein interaction studies to dissect signaling pathways and receptor-ligand dynamics.

• High Purity and Activity: Recombinant CD28 proteins are typically produced in mammalian systems (e.g., HEK293 cells) to ensure correct folding, post-translational modifications, and high bioactivity, as verified by binding assays and structural analyses.

• Translational Relevance: Using recombinant CD28 from human and non-human primates allows for the development and validation of immunotherapies, vaccines, and biologics in models that closely recapitulate human immune function, reducing translational gaps between animal studies and clinical applications.

• Batch Consistency and Experimental Control: Recombinant proteins provide reproducible results and eliminate variability inherent to primary cell or tissue-derived proteins, supporting robust experimental design.

In summary, recombinant Human/Cynomolgus/Rhesus Macaque CD28 is a versatile tool for dissecting T cell biology, developing immunotherapies, and bridging preclinical and clinical research in immunology.

Yes, you can use recombinant Human/Cynomolgus/Rhesus Macaque CD28 protein as a standard for quantification or calibration in ELISA assays, provided it is highly purified and its concentration is accurately determined. This approach is widely accepted for quantitative ELISA, especially when natural protein standards are unavailable.

Essential context and supporting details:

• Purity and Characterization: The recombinant CD28 protein should be of high purity (typically >90–95% by SDS-PAGE or SEC-HPLC). Confirm the protein’s identity and purity using appropriate analytical methods before use.

• Concentration Determination: Accurate quantification of the recombinant protein is critical. Use spectrophotometric methods (e.g., absorbance at 280 nm) or amino acid analysis to determine concentration.

• Standard Curve Preparation: Prepare a serial dilution of the recombinant CD28 in the same buffer or matrix as your samples to generate a standard curve. The concentration range should bracket the expected sample concentrations, typically from low pg/mL to ng/mL, depending on assay sensitivity.

• Matrix Effects: If your samples are in serum, plasma, or other complex matrices, spike the recombinant standard into the same matrix to account for potential matrix effects and ensure accurate quantification.

• Species Cross-reactivity: Since the recombinant protein includes sequences from human, cynomolgus, and rhesus macaque, confirm that your ELISA antibodies recognize the relevant epitopes. Most commercial anti-CD28 antibodies are designed to detect human CD28 and may cross-react with non-human primate CD28, but this should be validated for your assay.

• Documentation and Validation: Document the source, lot, and characterization data for the recombinant standard. Validate its performance in your ELISA by assessing recovery, linearity, and reproducibility.

Best practices:

• Use freshly reconstituted or properly stored recombinant protein to avoid degradation.
• Include blank and matrix-matched controls in each assay run.
• Validate the standard curve for each new lot of recombinant protein.

Limitations:

• Recombinant proteins may differ in glycosylation or folding compared to native proteins, potentially affecting antibody recognition or assay calibration. Always validate the standard in your specific ELISA system.
• If your ELISA is designed for human CD28 only, ensure the recombinant standard’s sequence and structure are compatible with the assay’s antibodies.

Summary Table: Recombinant CD28 as ELISA Standard

In conclusion: Recombinant Human/Cynomolgus/Rhesus Macaque CD28 is suitable as an ELISA standard if properly characterized, quantified, and validated for your specific assay system.

Recombinant Human/Cynomolgus/Rhesus Macaque CD28 has been validated in published research for several key applications, primarily related to immunological assays and cell-based functional studies involving T cell activation and co-stimulation.

Validated Applications in Published Research:

• T Cell Activation and Proliferation Assays:
  Recombinant CD28 is frequently used to provide the essential "signal 2" for T cell activation in vitro, often in combination with anti-CD3 antibodies. This co-stimulation is critical for robust T cell proliferation, cytokine production (such as IL-2, IFN-γ, and TNF-α), and prevention of T cell exhaustion. For example, in published studies, recombinant CD28 was used to coat plates or as a soluble factor to enhance T cell responses in human peripheral blood mononuclear cell (PBMC) cultures.

• Cytotoxicity and Tumor Cell Killing Assays:
  In the context of cancer immunotherapy research, recombinant CD28 has been used to enhance the cytotoxic activity of T cells against tumor cells, particularly when combined with CD3 bispecific antibodies (such as BiTEs). This combination leads to increased target cell lysis and T cell proliferation, supporting its use in evaluating immunotherapeutic strategies.

• ELISA and Protein-Protein Interaction Studies:
  Recombinant CD28 is used as a standard or capture protein in ELISA assays to study CD28 interactions, antibody binding, or to quantify CD28 levels. It is also employed in binding assays to characterize the affinity and specificity of anti-CD28 antibodies or ligands.

• Chimeric Antigen Receptor (CAR) T Cell Research:
  The CD28 domain is a common co-stimulatory element in CAR constructs. Recombinant CD28 proteins are used in the development and validation of CAR T cells, including functional assays to assess CAR signaling and T cell activation.

• Regulatory T Cell (Treg) Expansion:
  Published research indicates that CD28 co-stimulation is required for the expansion and maintenance of regulatory T cells (Tregs) in vitro and in vivo, making recombinant CD28 a tool for Treg functional studies.

Additional Context:

• Species Cross-Reactivity:
  The recombinant protein validated for human, cynomolgus, and rhesus macaque CD28 enables translational studies in non-human primate models, which are important for preclinical evaluation of immunotherapies.

• Assay Formats:
  Applications include plate-bound (immobilized) and soluble formats, depending on the experimental design. Plate-bound CD28 is often used for robust T cell activation, while soluble forms may be used for more physiological co-stimulation.

Summary Table of Validated Applications

These applications are supported by multiple peer-reviewed studies and product validation data, confirming the utility of recombinant CD28 in both basic and translational immunology research.

To reconstitute and prepare Recombinant Human/Cynomolgus/Rhesus Macaque CD28 protein for cell culture experiments, centrifuge the vial briefly before opening, then dissolve the lyophilized protein in sterile distilled water to a final concentration of 0.1–0.5 mg/mL. Avoid vortexing or vigorous pipetting to prevent protein denaturation.

Essential steps and best practices:

• Centrifuge the vial before opening to ensure all lyophilized material is at the bottom.
• Reconstitution: Add sterile distilled water gently to achieve the desired concentration (typically 0.1–0.5 mg/mL). Mix by gentle pipetting or slow inversion.
• Avoid harsh handling: Do not vortex or pipette vigorously, as this may denature the protein.
• Carrier protein/stabilizer: For long-term storage or to enhance stability, add a carrier protein or stabilizer such as 0.1% BSA, 5% HSA, 10% FBS, or 5% trehalose after reconstitution.
• Aliquoting: Divide the reconstituted solution into small aliquots to minimize freeze-thaw cycles.
• Storage: Store lyophilized protein at −20°C to −80°C. After reconstitution, keep aliquots at −20°C for up to 3 months, or at 2–8°C for up to 1 week for short-term use.
• Endotoxin: Confirm that endotoxin levels are suitable for cell culture (typically <1 EU/μg).

Preparation for cell culture:

• Use sterile technique throughout to prevent contamination.
• If required for functional assays, dilute the reconstituted protein further in cell culture medium or PBS, ensuring compatibility with your experimental system.
• For coating plates or stimulation assays, optimize concentration based on your specific protocol and cell type.

Summary Table:

Always consult the Certificate of Analysis for lot-specific instructions and confirm compatibility with your experimental design.