Recombinant Human CD86 (B7-2)

Recombinant Human CD86 is widely used in research to study immune cell activation, T cell costimulation, regulatory T cell (Treg) biology, and as a tool for immunotherapy target validation. Its use is justified by several key scientific applications:

• T Cell Costimulation and Activation: CD86 is a critical ligand for CD28 and CTLA-4 on T cells, providing essential costimulatory signals required for full T cell activation, proliferation, and cytokine production. Recombinant CD86 enables controlled in vitro studies of these pathways, allowing precise dissection of costimulatory requirements in human or model systems.

• Regulatory T Cell (Treg) Expansion and Function: CD86 has been shown to more effectively support the proliferation and survival of FoxP3+ Tregs compared to CD80, via CD28-mediated costimulation. This makes recombinant CD86 particularly valuable for studies focused on Treg biology, immune tolerance, and autoimmunity.

• Immunotherapy Target Validation: CD86 is an immune checkpoint molecule and a potential target for cancer immunotherapy. Its expression correlates with immune cell infiltration and prognosis in certain cancers, such as bladder cancer. Recombinant CD86 can be used to screen for therapeutic antibodies, small molecules, or to validate immune-modulatory strategies.

• Antigen Presentation and Dendritic Cell Maturation: CD86 is a marker of dendritic cell (DC) maturation and is involved in the regulation of antigen presentation. Recombinant CD86 can be used to study DC-T cell interactions, DC maturation, and the molecular mechanisms of immune synapse formation.

• Mechanistic Studies of Immune Regulation: Recombinant CD86 allows investigation of post-translational modifications (e.g., ubiquitination), ligand-receptor interactions, and the differential roles of CD80 vs. CD86 in immune regulation.

• Assay Development: Recombinant CD86 is used as a standard or capture reagent in ELISA, flow cytometry, and cell-based assays to quantify or detect CD86 interactions, or to stimulate cells in a defined manner.

Additional context:

• CD86 is expressed earlier than CD80 during immune responses and is involved in immunoglobulin class-switching.
• Recombinant proteins provide batch-to-batch consistency, defined activity, and eliminate variability associated with primary cell-derived material, making them ideal for reproducible research.

In summary, using recombinant human CD86 in research provides a controlled, reproducible tool to dissect immune costimulation, study Treg biology, validate immunotherapy targets, and develop robust immunological assays.

Yes, you can use recombinant human CD86 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 protocols for CD86, as most commercial ELISA kits are validated to detect both natural and recombinant forms of human CD86.

Key considerations and supporting details:

• ELISA kits for human CD86 are designed to recognize both endogenous (natural) and recombinant CD86. Multiple kit protocols explicitly state that their assays will detect and quantify both forms, making recombinant CD86 suitable as a standard.
• Recombinant proteins are widely used as ELISA standards because they can be produced in large quantities with defined purity and concentration, allowing for reproducible standard curves.
• Ensure the recombinant CD86 is well-characterized: The protein should be highly purified, and its concentration should be accurately determined (e.g., by absorbance at 280 nm or BCA assay). The standard curve should be prepared using serial dilutions in the same buffer as your samples to minimize matrix effects.
• Check compatibility with your assay: If you are using a commercial ELISA kit, verify that the recombinant CD86 matches the region (epitope) recognized by the kit’s capture and detection antibodies. Most kits are validated for full-length or extracellular domain recombinant proteins, but it is best to confirm this in the kit documentation or with the supplier.
• Carrier-free formulations are preferred for use as ELISA standards, as carrier proteins (e.g., BSA) can interfere with quantification or introduce variability.

Limitations:

• Some recombinant proteins are specifically labeled for use as ELISA standards, while others are not tested for this application and may not be suitable for quantitative calibration. Always check the product documentation for intended use.
• If your recombinant CD86 contains tags (e.g., His-tag), ensure these do not interfere with antibody binding in your ELISA format.

Best practice:
Prepare a standard curve using serial dilutions of your recombinant CD86 in the same matrix as your samples, and run it in parallel with your unknowns for accurate quantification.

Summary Table: Recombinant CD86 as ELISA Standard

In conclusion:
Recombinant human CD86 is suitable as a standard for ELISA quantification if it is pure, accurately quantified, and compatible with your assay system. Always consult your specific kit’s documentation for any additional requirements.

Recombinant Human CD86 has been validated for several key applications in published research, primarily in immunology and cell biology. The most commonly reported applications include:

• Flow Cytometry: Recombinant CD86 is used to assess binding interactions with its receptors (CD28, CTLA-4) on cell surfaces, and to characterize cell populations expressing CD86.
• ELISA (Enzyme-Linked Immunosorbent Assay): It serves as a standard or capture antigen to quantify soluble CD86 or its binding partners in biological samples.
• Cell Activation Assays: CD86 is used to stimulate T cells via CD28, leading to enhanced activation, proliferation, and cytokine production in vitro.
• Protein-Protein Interaction Studies: Immobilized recombinant CD86 is employed to study binding kinetics and affinities with CTLA-4, CD28, and other immune checkpoint molecules.
• Immunotherapy Research: Recombinant CD86 is used in preclinical models to investigate its role as a therapeutic target, particularly in classic Hodgkin lymphoma (cHL) and in the development of CAR T-cell therapies.
• Immunological Marker Studies: Soluble forms of CD86 are measured as biomarkers in disease states, such as abdominal aortic aneurysm.

Additional validated uses include:

• Western Blotting: Detection of CD86 protein in cell lysates.
• Adjuvant Mechanism Studies: Evaluating the requirement of CD86 in immune cell activation and adjuvant efficacy.

These applications leverage recombinant CD86’s ability to mimic native protein interactions, enabling detailed study of immune co-stimulation, checkpoint inhibition, and therapeutic targeting in both basic and translational research contexts.

To reconstitute and prepare Recombinant Human CD86 protein for cell culture experiments, follow these general guidelines based on manufacturer protocols and best practices:

1. Reconstitution

• For lyophilized protein:

  • Centrifuge the vial briefly before opening to ensure the powder is at the bottom.
  • Reconstitute with sterile, endotoxin-free PBS or a serum-free cell culture medium.
  • Typical reconstitution concentration: 0.1–1.0 mg/mL.
    • Example: For 100 µg of protein, add 100–1000 µL of buffer to achieve 1–0.1 mg/mL.
  • Gently swirl or rotate the vial to dissolve the protein. Avoid vigorous shaking to prevent foaming and denaturation.
  • Allow the protein to reconstitute for 15–30 minutes at room temperature with gentle agitation.

• For liquid protein:

  • If the protein is supplied in liquid form, simply dilute it to the desired working concentration using sterile PBS or serum-free medium.

2. Storage of Stock Solution

• Aliquot the reconstituted protein to avoid repeated freeze-thaw cycles.
• Store aliquots at –20°C or –80°C for long-term storage.
• For short-term use, store at 2–8°C for up to one week.

3. Preparation for Cell Culture

• Coating plates (for T cell activation or interaction studies):
  • Dilute the reconstituted CD86 protein in PBS or serum-free medium to a final concentration of 1–10 µg/mL.
  • Add the diluted protein to the culture surface (e.g., 1–2 µg/well for a 6-well plate).
  • Incubate at room temperature for 1–2 hours.
  • Aspirate the solution and rinse the plate gently with sterile water or PBS.
  • The coated plate is now ready for cell culture or can be stored at 2–8°C if kept sterile.

4. Handling and Stability

• Avoid repeated freeze-thaw cycles.
• Use sterile technique throughout to prevent contamination.
• If needed, add carrier proteins (e.g., 0.2–1% BSA or HSA) to stabilize the protein in solution.

5. Activity and Quality Control

• Confirm protein activity using functional assays (e.g., binding to CTLA-4 or CD28).
• Check for endotoxin levels if using for sensitive cell culture applications.

These steps ensure proper reconstitution and preparation of Recombinant Human CD86 for reliable results in cell culture experiments. Always refer to the specific product datasheet for any unique requirements.