Recombinant Human BCA-1

Recombinant Human BCA-1 (CXCL13) is primarily used in research applications to study B cell migration, lymphoid tissue organization, and immune cell chemotaxis due to its potent and selective chemoattractant activity for B lymphocytes.

Key scientific reasons to use Recombinant Human BCA-1 in research:

• B cell migration assays: BCA-1 is a highly specific chemoattractant for B lymphocytes, acting through the CXCR5 receptor (formerly BLR1), which is expressed on mature B cells and a subset of T helper memory cells. This makes it essential for in vitro and in vivo studies of B cell chemotaxis, homing, and follicular organization.

• Lymphoid tissue organization: BCA-1 is highly expressed in secondary lymphoid organs (spleen, lymph nodes, Peyer's patches) and is critical for the proper formation of B cell follicles and germinal centers. Disruption of the CXCL13/CXCR5 axis impairs lymphoid architecture, making recombinant BCA-1 valuable for mechanistic studies of lymphoid tissue development and maintenance.

• Immune response and inflammation: CXCL13/BCA-1 is implicated in chronic inflammation, lymphoid neogenesis, and autoimmune pathogenesis. Recombinant BCA-1 can be used to model these processes, investigate signaling pathways, and study its role in disease states.

• Functional bioassays: Recombinant BCA-1 is biologically active and suitable for chemotaxis assays, GTPγS binding assays, and calcium flux studies in cells expressing CXCR5 or CXCR3. It is also used to validate antibody specificity and function in ELISA and Western blotting.

• Human relevance: Recombinant human BCA-1 allows for direct study of human immune cell behavior, avoiding species-specific differences seen with murine CXCL13.

Additional technical considerations:

• Recombinant BCA-1 is typically produced as a non-glycosylated polypeptide in E. coli, with a molecular mass of ~10–13 kDa, and is available with or without affinity tags for purification and detection.
• It is suitable for use in cell culture, migration assays, and as a standard in immunoassays.

Summary of applications:

• B cell chemotaxis and migration studies
• Lymphoid tissue development and organization research
• Autoimmunity and chronic inflammation models
• Functional assays for chemokine receptor signaling
• Immunoassay standardization and antibody validation

Using recombinant human BCA-1 enables precise, reproducible, and human-relevant investigation of B cell biology and immune system function in both basic and translational research contexts.

Yes, you can use recombinant human BCA-1 as a standard for quantification and calibration in ELISA assays, with important considerations regarding cross-reactivity and assay design.

Suitability as an ELISA Standard

Recombinant human BCA-1 (CXCL13) is suitable for use as a calibration standard in sandwich ELISA formats. The recombinant protein has been demonstrated to accurately quantitate itself when used as a standard, and results obtained using natural human BCA-1 show linear curves that are parallel to standard curves generated with recombinant BCA-1 standards. This parallel behavior indicates that the recombinant protein behaves similarly to the native form in the immunoassay system.

Cross-Reactivity Considerations

A critical distinction exists between assay design intentions and actual performance. While some ELISA kits are specifically designed to detect native BCA-1 rather than recombinant forms, the antibodies used in sandwich ELISA systems typically recognize both recombinant and naturally produced human BCA-1 proteins. This dual recognition capability means that recombinant BCA-1 can serve as an effective standard for measuring both endogenous and recombinant forms of the protein in your samples.

Standard Preparation Best Practices

When using recombinant BCA-1 as your calibration standard, follow these guidelines:

Reconstitution: Most recombinant protein standards are provided in lyophilized form and require reconstitution. Carefully follow the lot-specific reconstitution instructions provided with your standard, as protocols may vary between batches.

Concentration Verification: If you prepare your own standards from recombinant protein, verify the protein concentration using appropriate quantification methods such as HPLC or spectrophotometric assays before establishing your calibration curve.

Standard Curve Development: Prepare serial dilutions of your recombinant BCA-1 standard across the expected assay range. For human BCA-1 ELISA applications, typical measurable ranges span from approximately 1-1000 pg/mL, depending on your specific assay platform and antibody pair.

Performance Validation

To validate that your recombinant BCA-1 standard performs appropriately, assess the linearity of your standard curve and confirm that recovery studies with spiked samples yield acceptable results (typically 85-115% recovery). This validation ensures that your calibration standard accurately reflects the behavior of BCA-1 in your specific assay matrix.

Recombinant Human BCA-1 (CXCL13) has been validated for several key applications in published research, primarily centered around its role as a chemoattractant and its involvement in immune cell migration and signaling. The main applications include:

• Bioassay: Recombinant Human BCA-1 is widely used in bioassays to study its chemoattractant properties, particularly for B lymphocytes. It has been validated for use in functional assays to assess cell migration, homing, and activation of B cells and a subset of memory T cells. These assays are typically performed using whole cells, including primary human lymphocytes and cell lines.

• ELISA: Recombinant BCA-1 is used as a standard in ELISA kits to quantify CXCL13 levels in various biological samples such as serum, plasma, cell culture supernates, and cerebrospinal fluid (CSF). This application is critical for biomarker studies and monitoring disease activity in conditions like HIV, rheumatoid arthritis, and lymphoma.

• Functional Studies: The protein is employed in functional studies to investigate the role of CXCL13 in germinal center formation, B cell trafficking, and immune responses. These studies often involve in vitro and in vivo models to assess the impact of CXCL13 on immune cell behavior and disease progression.

• Protein Array and SDS-PAGE: Recombinant BCA-1 is used in protein array and SDS-PAGE applications for protein characterization and interaction studies.

These applications highlight the versatility of Recombinant Human BCA-1 in both basic research and clinical studies, particularly in immunology and oncology.

To properly reconstitute and prepare Recombinant Human BCA-1 (CXCL13) protein for cell culture experiments, follow these best practices based on manufacturer guidelines and scientific protocols:

Reconstitution Steps:

1. Centrifuge the Vial:
   Before opening, briefly centrifuge the lyophilized protein vial to ensure all powder is at the bottom.

2. Reconstitution Buffer:
   Reconstitute the protein in sterile PBS (phosphate-buffered saline) or sterile water, as specified by the manufacturer.

   • Typical reconstitution concentration: 25–100 μg/mL (check the product datasheet for the recommended concentration).
   • For example, for 100 μg of protein, add 1–4 mL of buffer to achieve 25–100 μg/mL.

3. Gentle Mixing:
   Gently pipette the buffer down the side of the vial.

   • Do not vortex or shake vigorously to avoid protein denaturation.
   • Allow the vial to sit at room temperature for 15–30 minutes with gentle agitation (e.g., slow rocking) to fully dissolve the protein.

4. Add Carrier Protein (Optional but Recommended):
   For increased stability, especially if storing aliquots, add a carrier protein such as 0.1% BSA (bovine serum albumin) or 0.1% HSA (human serum albumin) to the reconstituted solution.

Preparation for Cell Culture:

1. Aliquot and Store:

   • Aliquot the reconstituted protein into small volumes to avoid repeated freeze-thaw cycles.
   • For short-term use (≤1 week), store at 2–8°C under sterile conditions.
   • For long-term storage, freeze aliquots at –20°C to –80°C.

2. Dilution for Cell Culture:

   • Dilute the reconstituted protein in cell culture medium or assay buffer to the desired working concentration (typically 10–100 ng/mL, depending on your experiment).
   • If using serum-free medium, avoid carrier proteins like BSA; instead, use stabilizers like trehalose if needed.

3. Avoid Repeated Freeze-Thaw Cycles:
   Thaw aliquots only once and discard any unused portion after thawing.

Summary of Key Points:

• Reconstitution: Sterile PBS or water, gentle mixing, no vortexing.
• Carrier Protein: Add 0.1% BSA/HSA for stability (optional for serum-free cultures).
• Storage: Aliquot, store at 2–8°C for ≤1 week or –20°C to –80°C for long-term.
• Working Dilution: Dilute in culture medium to desired concentration (e.g., 10–100 ng/mL).
• Avoid: Repeated freeze-thaw cycles, vigorous shaking.

Always refer to the Certificate of Analysis (CoA) or product datasheet for specific instructions, as protocols may vary slightly between manufacturers.