Recombinant Human CD6

Recombinant Human CD6 is a valuable tool in research applications focused on immunology, autoimmunity, and cancer due to its central role in T cell activation, costimulation, and cell adhesion. Using recombinant CD6 enables precise mechanistic studies, therapeutic screening, and functional assays relevant to both disease modeling and drug development.

Key reasons to use Recombinant Human CD6 in research:

• Modeling T Cell Activation and Costimulation: CD6 is a surface glycoprotein expressed on T cells, where it modulates activation, proliferation, and adhesion through interactions with its ligand ALCAM (CD166). Recombinant CD6 can be used in vitro to dissect these pathways, study T cell responses, and evaluate the impact of CD6-ALCAM interactions on immune synapse formation and signaling.

• Autoimmunity Research: CD6 is implicated in the pathogenesis of several autoimmune diseases, including multiple sclerosis and uveitis. Recombinant CD6 can be used to:

  • Block endogenous CD6-ligand interactions, thereby modulating T cell activation and differentiation.
  • Serve as a decoy receptor to mask CD6 ligands in vivo, which has shown modest anti-inflammatory and anti-cancer effects in animal models.
  • Screen and characterize therapeutic antibodies or small molecules targeting CD6 for immunosuppressive or immunomodulatory effects.

• Cancer Immunotherapy: CD6 is emerging as a target for cancer immunotherapy because of its dual role in regulating cytotoxic lymphocyte activity and suppressing autoimmunity. Recombinant CD6 can be used to:

  • Investigate the mechanisms by which CD6 modulates NK and CD8+ T cell cytotoxicity against tumor cells.
  • Evaluate the effects of CD6-targeted therapies on tumor cell killing and immune cell infiltration in preclinical models.
  • Study the balance between anti-tumor immunity and the risk of autoimmunity when manipulating CD6 pathways.

• Therapeutic Antibody Development: Recombinant CD6 is essential for screening, binding studies, and functional assays involving anti-CD6 monoclonal antibodies, which are being developed for both immunosuppression (e.g., in transplantation) and immunostimulation (e.g., in cancer).

• Cell Adhesion and Migration Studies: CD6 mediates T cell adhesion to antigen-presenting cells and endothelium, influencing immune cell trafficking and tissue infiltration. Recombinant CD6 can be used in cell adhesion assays and to study the molecular basis of immune cell migration.

• Versatility in Experimental Design: Recombinant CD6 is available in various formats (e.g., Fc chimeras, His-tagged proteins), allowing flexibility for use in ELISA, flow cytometry, cell-based assays, and in vivo studies.

Summary of Applications:

• Mechanistic studies of T cell activation and costimulation
• Blocking or mimicking CD6-ligand interactions in vitro and in vivo
• Screening and characterization of therapeutic antibodies or small molecules
• Modeling autoimmune and cancer immunotherapy mechanisms
• Cell adhesion and migration assays

In summary, recombinant human CD6 is a critical reagent for dissecting immune regulatory mechanisms, developing novel immunotherapies, and modeling disease processes where T cell function is central.

Yes, recombinant human CD6 can be used as a standard for quantification or calibration in ELISA assays, provided it is of sufficient purity and its concentration is accurately determined. This is a common practice in quantitative ELISA protocols for measuring CD6 in biological samples.

Essential context and supporting details:

• Recombinant proteins as ELISA standards: Recombinant human CD6 is routinely used as a standard in commercial ELISA kits for quantification of CD6 in serum, plasma, tissue homogenates, and cell culture supernatants. The standard curve is generated by serially diluting the recombinant CD6 and measuring its signal, which allows interpolation of sample concentrations.

• Purity and quantification: For accurate calibration, the recombinant CD6 should be highly purified, and its concentration must be precisely known, typically determined by absorbance at 280 nm, BCA assay, or HPLC. Impurities or inaccurate quantification can lead to errors in the standard curve and sample measurement.

• Matrix effects: When using recombinant CD6 as a standard, it is important to prepare standards in a buffer that closely matches the sample matrix (e.g., serum, plasma, or cell culture medium) to minimize matrix effects and ensure comparable binding and detection.

• Validation: Commercial ELISA kits validate their recombinant CD6 standards for recovery, linearity, and specificity in various matrices. If using a custom recombinant CD6, similar validation steps should be performed to confirm its suitability as a standard.

• Limitations: Recombinant CD6 used as an ELISA standard is not recommended for bioassay applications unless specifically validated for those purposes. For quantification, ensure the recombinant protein is compatible with the antibodies and detection system used in your assay.

Best practices:

• Use recombinant human CD6 that is well-characterized and free of contaminants.
• Prepare a standard curve with serial dilutions covering the expected concentration range in your samples.
• Run the standard curve with each assay to account for day-to-day and batch-to-batch variability.
• Validate recovery and linearity in your specific sample matrix if possible.

Summary Table: Recombinant Human CD6 as ELISA Standard

In conclusion, recombinant human CD6 is suitable as a standard for ELISA quantification, provided it meets purity, quantification, and validation requirements.

Recombinant Human CD6 has been validated for several key applications in published research, primarily in immunology and cancer biology. The main validated uses include:

• In vitro functional assays: Recombinant soluble human CD6 (sCD6) has been used to inhibit T-cell proliferation, modulate T-cell activation, and study receptor–ligand interactions, particularly with ALCAM (CD166).
• In vivo therapeutic studies: Recombinant sCD6 has been administered to mice to assess its effects on tumor growth, metastasis, and immune regulation, demonstrating delayed tumor progression and altered regulatory T cell function.
• Autoimmune disease models: Recombinant human CD6 has been used in humanized mouse models to study its role in multiple sclerosis and other T-cell–driven autoimmune conditions, including experimental autoimmune encephalomyelitis (EAE).
• Cell adhesion and migration assays: CD6 has been used to investigate its adhesive properties and its role in cell–cell interactions, especially in the context of immune synapse formation and T cell migration.
• Immunoprecipitation, ELISA, Western blot (WB), and flow cytometry (FC): Recombinant CD6 protein has been validated for use as a standard or capture antigen in ELISA, as a positive control in WB, and for detection or blocking in FC and immunoprecipitation protocols.

Supporting details:

• In cancer research, recombinant sCD6 acts as a decoy receptor, interfering with CD6–ALCAM interactions, reducing tumor cell proliferation and migration, and modulating T cell effector functions.
• In autoimmune disease models, recombinant human CD6 has been used to test the efficacy of anti-CD6 monoclonal antibodies and to study the molecular mechanisms of T cell regulation.
• Recombinant CD6 is also used to remove CD6+ T cells from donor bone marrow prior to transplantation, highlighting its utility in cell separation protocols.
• Published protocols confirm its use in standard immunoassays (ELISA, WB, FC), immunoprecipitation, and as a tool for studying CD6-mediated signaling and adhesion.

Summary of validated applications:

• Functional immunology assays (in vitro and in vivo)
• Cancer and autoimmune disease models
• Cell adhesion and migration studies
• Immunoassays (ELISA, WB, FC, immunoprecipitation)
• Cell separation protocols

These applications are supported by multiple peer-reviewed studies and are widely used in research focused on T cell biology, immune regulation, and therapeutic development.

To reconstitute and prepare Recombinant Human CD6 protein for cell culture experiments, follow these steps for optimal solubility, stability, and biological activity:

Reconstitution Protocol:

• Centrifuge the vial briefly before opening to ensure all lyophilized material is at the bottom.
• Add sterile distilled water to achieve a final concentration of 0.1–0.5 mg/mL (typical stock concentration).
• Gently mix by tilting or slow pipetting. Avoid vortexing or vigorous pipetting, as this may denature the protein.
• Allow the protein to dissolve for 15–30 minutes at room temperature. If undissolved particles remain, gently mix for up to 2 hours.

Stabilization and Storage:

• For long-term storage, add a carrier protein or stabilizer such as 0.1% BSA, 5% HSA, 10% FBS, or 5% Trehalose to the reconstituted solution.
• Aliquot the solution to minimize freeze-thaw cycles.
• Store aliquots at -20°C for up to 3 months or at 2–8°C for up to 1 week.
• Lyophilized protein is stable at -20°C to -80°C for up to 1 year.

Preparation for Cell Culture:

• Before use, dilute the stock solution in sterile, cell culture-grade buffer (e.g., PBS or culture medium) to the desired working concentration, typically in the μg/mL range depending on your assay requirements.
• If using for coating plates (e.g., adhesion assays), a concentration of 5 μg/mL is commonly used.
• Ensure the final solution is endotoxin-free (recommended: <0.1 EU/μg for sensitive cell assays).

General Best Practices:

• Always use aseptic technique to prevent contamination.
• Wear appropriate personal protective equipment (lab coat, gloves).
• Avoid repeated freeze-thaw cycles to preserve protein integrity.

Summary Table:

These steps ensure the Recombinant Human CD6 protein is properly reconstituted and prepared for reliable cell culture experiments.