Recombinant Human Eotaxin-3

Recombinant Human Eotaxin-3 (CCL26) is used in research applications primarily to study the mechanisms of eosinophil and basophil recruitment, inflammatory responses, and diseases characterized by eosinophilia such as asthma, allergic disorders, and certain gastrointestinal conditions.

Key scientific applications and rationale:

• Chemotaxis Assays: Eotaxin-3 is a potent chemoattractant for eosinophils and basophils, acting through the CCR3 receptor. It is widely used to induce and quantify chemotactic responses in vitro, helping to elucidate the signaling pathways and cellular behaviors involved in immune cell migration.

• Receptor Activation Studies: Recombinant Eotaxin-3 enables detailed analysis of CCR3 activation, receptor-ligand specificity, and downstream signaling events. This is critical for understanding allergic inflammation and for screening potential antagonists or inhibitors for therapeutic development.

• Disease Modeling: Elevated Eotaxin-3 expression is associated with tissue eosinophilia and mastocytosis, notably in conditions such as eosinophilic esophagitis and asthma. Recombinant protein allows for mechanistic studies in cell culture and animal models, including investigation of genetic variants and their impact on disease susceptibility.

• Biomarker Development: Eotaxin-3 is being explored as a plasma biomarker for mucosal eosinophil infiltration, aiding in the diagnosis and monitoring of inflammatory diseases.

• Structure-Function Analysis: Recombinant Eotaxin-3, including mutant variants, is used to dissect the functional domains required for CCR3 binding and activation, informing drug design and basic immunology.

• Standardization in Bioassays: Recombinant Eotaxin-3 provides a consistent, high-purity standard for ELISA, binding assays, and other quantitative techniques, ensuring reproducibility and accuracy in experimental results.

Best practices:

• Use carrier-free formulations for binding and chemotaxis assays to avoid interference.
• Select appropriate concentrations (typically 0.3–2 µg/mL for chemotaxis) based on bioactivity data.
• Store and handle according to manufacturer’s recommendations to preserve protein integrity.

Summary of scientific value:
Using recombinant human Eotaxin-3 in research enables precise, reproducible investigation of eosinophil biology, allergic inflammation, and chemokine-receptor interactions, supporting both basic and translational studies in immunology and disease pathogenesis.

Yes, recombinant human Eotaxin-3 can be used as a standard for quantification or calibration in ELISA assays, provided it is properly validated for your specific assay format. Recombinant Eotaxin-3 is commonly supplied as a standard in commercial ELISA kits and is used to generate standard curves for quantitative measurement of Eotaxin-3 in biological samples.

Key considerations and best practices:

• Source and Purity: The recombinant Eotaxin-3 standard should be highly purified and its concentration accurately determined. Most commercial kits calibrate their assays against recombinant Eotaxin-3 expressed in E. coli.
• Matrix Compatibility: Ensure the recombinant standard is 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 assay detects both natural and recombinant Eotaxin-3 equivalently. Commercial ELISA kits report that standard curves generated with recombinant Eotaxin-3 are parallel to those obtained with natural Eotaxin-3, supporting its use for quantification.
• Reconstitution and Handling: Follow manufacturer or protocol instructions for reconstitution, dilution, and storage to maintain protein integrity and reproducibility.
• Assay Range: Prepare serial dilutions of the recombinant standard to cover the dynamic range of your assay, typically from low pg/mL to several ng/mL depending on sensitivity.

Limitations:

• Use recombinant Eotaxin-3 standards only for research purposes, not for diagnostic procedures, unless specifically validated for clinical use.
• Avoid mixing standards or reagents from different sources or lots, as this can introduce variability.

Summary Table: Use of Recombinant Human Eotaxin-3 as ELISA Standard

In summary, recombinant human Eotaxin-3 is suitable and widely used as a standard for ELISA quantification, provided you follow best practices for validation and assay setup.

Research Applications and Validation

Recombinant human Eotaxin-3 (CCL26) has been validated across multiple research applications, with particular emphasis on its chemotactic properties and role in inflammatory and allergic disease models.

Chemotaxis and Cell Migration Studies

The primary validated application involves chemotaxis assays, where recombinant Eotaxin-3 demonstrates potent activity in recruiting immune cells. Both the 71 amino acid and 68 amino acid variants have been shown to induce chemotaxis of human CCR3-transfected cell lines with equal potency. The protein induces calcium flux in eosinophils and CCR3-transfected cells, making it suitable for functional bioassays measuring cellular migration and activation.

Biomarker and Diagnostic Applications

Eotaxin-3 has been validated as a plasma biomarker for mucosal eosinophil infiltration, particularly in chronic rhinosinusitis with nasal polyps. Research demonstrates a significant positive correlation between plasma eotaxin-3 levels and mucosal eosinophil counts, establishing its utility in identifying patients with high eosinophilic infiltration who may require multiple surgical interventions. Additionally, eotaxin-3 expression has been validated as an optimal marker for defining type 2 inflammation in airway disease, correlating with IL-13-driven gene expression patterns in asthma patients.

Allergic and Eosinophilic Disease Models

The protein has been validated in studies examining allergic diseases and eosinophil-predominant disorders, including asthma, allergic rhinitis, atopic dermatitis, and eosinophilic esophagitis. Elevated serum eotaxin-3 levels have been documented in patients with urticaria compared to healthy controls, and gene expression levels are elevated in skin lesions of atopic dermatitis patients.

Therapeutic Response Assessment

Recombinant Eotaxin-3 has been validated for monitoring therapeutic efficacy, particularly in evaluating responses to dupilumab treatment. Serum eotaxin-3 levels decrease within 2 weeks of dupilumab administration, preceding clinical improvements in nasal polyp scores and sinonasal symptoms. This application supports its use in endotyping and predicting treatment outcomes in chronic rhinosinusitis.

Receptor Binding and Signaling Studies

The protein is validated for receptor binding assays and studies examining CCR3-mediated signaling pathways. Its ability to cross-desensitize cells to other CCR3 ligands has been documented, making it useful for investigating chemokine receptor interactions and signal transduction mechanisms.

Reconstitution Protocol

Recombinant Human Eotaxin-3 (CCL26) is supplied as a lyophilized powder and requires proper reconstitution before use in cell culture experiments. The reconstitution approach depends on the specific formulation variant you are using.

Standard Reconstitution Procedures

For formulations with carrier protein: Reconstitute at 25 μg/mL in sterile PBS containing at least 0.1% human or bovine serum albumin. This carrier protein is essential for maintaining protein stability and preventing non-specific adsorption to container surfaces.

For carrier-free formulations: Reconstitute at 100 μg/mL in sterile PBS. This variant is suitable when carrier proteins would interfere with downstream applications.

General reconstitution guidelines: Centrifuge the vial before opening to ensure all lyophilized material is at the bottom. Add the appropriate volume of sterile distilled water or PBS to achieve your desired concentration. Do not vortex—instead, gently pipette the solution up and down the sides of the vial to suspend the protein. Allow several minutes for complete dissolution.

Further Dilutions for Cell Culture

For working concentrations below the initial reconstitution level, prepare further dilutions in low endotoxin medium or buffered solutions containing carrier proteins such as heat-inactivated fetal calf serum or tissue culture-grade bovine serum albumin. This approach maintains protein integrity during serial dilutions and prevents loss of bioactivity.

Storage and Stability

Lyophilized protein: Store at -20°C in a manual defrost freezer. The lyophilized form remains stable for at least 2 years from the date of receipt.

Reconstituted protein: After aseptic reconstitution, store reconstituted Eotaxin-3 in working aliquots with a carrier protein at -20°C for up to six months, or at 2-8°C for up to one month. Avoid repeated freeze-thaw cycles, as these can compromise protein activity and lead to aggregation.

Quality Considerations

The lyophilized protein is typically formulated from a 0.2 μm filtered solution in acetonitrile and trifluoroacetic acid with BSA as a carrier protein. Endotoxin levels are generally maintained at less than 1.0 EU/μg as determined by LAL method, which is critical for cell culture applications where endotoxin contamination could confound experimental results.

Human Eotaxin-3 is chemotactic for eosinophils and PHA-activated T cells and induces calcium flux in eosinophils as well as in CCR3-transfected cells, making it suitable for various immunological and cell migration assays.