Anti-Human CXCR4 (Clone 12G5) – Purified in vivo GOLD™ Functional Grade

Use of Clone 12G5 (Anti-CXCR4/CD184) in In Vivo Mouse Studies

Summary
Clone 12G5 is a widely used monoclonal antibody targeting human CXCR4 (CD184), a chemokine receptor involved in cell migration, immune responses, and cancer biology. While most common uses of 12G5 are in vitro (e.g., flow cytometry, immunohistochemistry), it has also been employed in specialized in vivo mouse studies, particularly in oncology and imaging research.

Key In Vivo Applications

In Vivo Imaging and Biodistribution Studies
One of the most direct in vivo applications of clone 12G5 in mice involves its use as a radiolabeled ([125I]12G5) tracer to evaluate CXCR4 expression in brain tumor xenografts. Mice implanted with human tumors (U87 human glioblastoma) were intravenously injected with [125I]12G5, and tissue distribution was measured over several days. The study tracked antibody uptake in tumor versus normal tissues using gamma spectrometry and found that 12G5 accumulated preferentially in tumors compared to normal organs, reaching maximal tumor uptake at 48 hours post-injection. This approach demonstrated the feasibility of using 12G5 for in vivo immunoimaging and biodistribution in preclinical models, potentially guiding the development of CXCR4-targeted diagnostic or therapeutic agents.

Mechanism and Rationale
The rationale for using clone 12G5 in such studies is its specificity for human CXCR4, making it suitable for xenograft models where human cancer cells or tissue are implanted into immunocompromised (e.g., SCID or nude) mice. The antibody's ability to bind CXCR4 (and potentially modulate its function) allows researchers to visualize and quantify receptor expression in vivo, which is especially relevant in cancers where CXCR4 is associated with aggressive tumor growth, invasion, and metastasis.

Clinical and Translational Implications

Functional Blockade and Therapeutic Potential
While in vivo studies directly delivering 12G5 for functional blockade (e.g., inhibiting CXCR4-dependent cell migration or tumor growth) in mice are less commonly reported, the antibody has been shown in vitro to neutralize CXCR4 function, blocking SDF-1 (CXCL12)-induced chemotaxis and inhibiting HIV infection in CXCR4+ cells. The in vivo biodistribution data suggest that, if delivered systemically, 12G5 could potentially interact with CXCR4-expressing tumors, making it a candidate for further translational research in targeted therapies.

Technical Considerations

Species Specificity
Clone 12G5 is specific for human CXCR4 and does not cross-react with mouse CXCR4. Therefore, its use in mice is limited to xenograft models with human cells or tissues, and not in native murine cancer or immune studies.

Delivery and Monitoring

• Intravenous Injection: Radiolabeled 12G5 is typically administered via tail vein injection in mice.
• Imaging and Quantification: Tissue distribution and receptor expression are monitored using radioactivity measurements or, in principle, noninvasive imaging techniques.
• Pharmokinetics: Rapid clearance from the blood (likely due to deiodination and tissue uptake) has been observed, with spleen accumulation noted as the highest among normal organs.

Summary Table: In Vivo Applications of Clone 12G5 in Mice

Conclusion

Clone 12G5 is primarily used in in vivo mouse studies involving human tumor xenografts, where it serves as a specific probe for human CXCR4 expression and distribution via radiolabeled imaging. Its application is restricted to models with human cells due to species specificity. While it has shown promise in biodistribution and imaging studies, its direct therapeutic or functional blockade potential in vivo remains a subject for future investigation.

Storage Temperature for Sterile Packaged Clone 12G5

Optimal Storage Conditions

Clone 12G5 is a widely used CXCR4 monoclonal antibody. The correct storage temperature depends on the formulation and manufacturer’s instructions.

• For the Human CXCR4 Antibody MAB170 (lyophilized, from R&D Systems):
  As supplied (lyophilized, sterile, packaged), store at -20 °C to -70 °C for up to 12 months from the date of receipt.
  After reconstitution, the antibody is stable for 1 month at 2–8 °C (refrigerator) or 6 months at -20 °C to -70 °C (freezer).
• For Anti-CXCR4 Mouse Monoclonal Antibody (biotin or fluorescein labeled, pre-diluted in buffer):
  Do not freeze. Store at 2–8 °C (refrigerator) for up to 12 months from the date of receipt.

Key Considerations

• Always check the manufacturer’s instructions and product label for the specific clone/conjugate/format you are using, as storage conditions may vary by supplier.
• For lyophilized (powder) formulations, long-term storage is best at -20 °C or lower until reconstituted, after which refrigerated or re-frozen storage (but not repeated freeze-thaw cycles) is acceptable per manufacturer guidance.
• For liquid, pre-diluted formulations, do not freeze—store at 2–8 °C only and protect from light.
• For sterile packaged products, maintaining sterility means using proper aseptic technique for opening, aliquoting, and storing.

Summary Table

Always follow the product-specific guidelines provided with your antibody clone 12G5 for best stability and performance.

Alongside 12G5, which targets CXCR4 (CD184), the literature commonly pairs it with other antibodies or proteins that recognize CCR5 (CD195) and their ligands, especially in studies related to HIV entry, chemokine receptor biology, and immune cell subset identification.

Frequently used in combination with 12G5 are:

• 2D7: A monoclonal antibody specific for CCR5, used for flow cytometry, blocking studies, and mapping CCR5 expression. 2D7 serves as the CCR5 counterpart to 12G5 for CXCR4 and is commonly cited in HIV-1 entry inhibition and chemokine receptor research.
• PRO 140: A humanized antibody targeting CCR5 with clinical applications in HIV-1 therapy. It is often used alongside 12G5 and 2D7 in competitive binding, inhibition, or immunophenotyping panels, because it recognizes a conformational epitope on CCR5.
• CD4 antibodies: Since CD4 works as the primary receptor for HIV entry, anti-CD4 antibodies are frequently included to distinguish the roles of CD4 versus CXCR4 or CCR5 in viral entry and cell signaling.
• SDF-1 (CXCL12): Although not an antibody, the CXCR4 ligand SDF-1 is often used in conjunction with 12G5 to assess receptor function, blocking, and neutralization efficacy in migration and signaling assays.
• Isotype control antibodies: Used as specificity controls in flow cytometry and blocking experiments with 12G5.

These antibodies and proteins together allow:

• Comprehensive phenotyping of immune cells expressing CXCR4 and CCR5
• Functional studies of chemokine-mediated migration, signaling, and HIV inhibition
• Assessment of receptor blocking or activation in both research and clinical settings.

Additional combinations can depend on cell type or context (e.g., B cell, T cell, or stem cell markers), but 2D7 and PRO 140 are the most consistently co-cited protein reagents with 12G5 in immunology and HIV literature.

The monoclonal antibody 12G5 is a significant tool in scientific research, particularly noted for its specificity against the human CXCR4 receptor. Here are the key findings from scientific literature regarding clone 12G5:

1. CXCR4 Specificity: The antibody 12G5 specifically binds to the CXCR4 receptor, which is a G-protein-coupled chemokine receptor involved in various cellular processes, including cell migration and signaling.

2. HIV Research: In the context of HIV, 12G5 blocks the fusion of certain HIV strains with host cells by inhibiting the interaction between HIV and the CXCR4 co-receptor. It effectively inhibits infections by HIV-1 strains that utilize CXCR4, such as HIV-1(89.6), but has little effect on strains like HIV-1(IIIB).

3. Antagonist Competition: The 12G5 antibody competes with CXCR4 antagonists for binding sites on the receptor, particularly in the second extracellular loop (ECL2). This is crucial for understanding how different compounds interact with CXCR4 and for developing new therapeutic strategies.

4. Biological Functions: CXCR4 plays a role in homing hematopoietic stem cells and mobilizing them in response to stress or injury. It is also implicated in various pathologies, including autoimmune diseases and cancer.

5. Experimental Use: Clone 12G5 has been utilized in flow cytometry experiments to study CD184 (CXCR4) expression on cell surfaces. This has helped in understanding the potential impact of proteins like IFN-induced transmembrane proteins on viral infections.

In summary, 12G5 is a valuable tool for studying CXCR4-mediated processes, particularly in the context of HIV infection and cell signaling, and provides insights into the interaction between CXCR4 and its antagonists.