Development of Sensitive Anti-Mouse CCR5 Monoclonal Antibodies Using the N-Terminal Peptide Immunization

Area of Science:

• Immunology
• Molecular Biology
• Biochemistry

Background:

• C-C chemokine receptor 5 (CCR5) is a G protein-coupled receptor crucial for immune cell activation and migration.
• CCR5 signaling regulates innate and adaptive immunity, making it a therapeutic target for inflammatory diseases and tumors.
• Existing anti-CCR5 monoclonal antibodies (mAbs) have been explored in clinical trials.

Purpose of the Study:

• To develop and characterize novel monoclonal antibodies (mAbs) targeting mouse CCR5 (mCCR5).
• To evaluate the utility of these novel mAbs for detecting mCCR5 in various experimental settings.

Main Methods:

• N-terminal peptide immunization was used to generate anti-mCCR5 mAbs.
• Flow cytometry was employed to assess mAb binding to mCCR5-expressing cell lines (CHO/mCCR5 and L1210).
• Western blotting was performed to confirm mCCR5 detection by the generated mAbs.

Main Results:

• Two novel anti-mCCR5 mAbs, C5Mab-4 (rat IgG2a) and C5Mab-8 (rat IgG1), were successfully established.
• Both mAbs demonstrated specific binding to mCCR5-expressing cells via flow cytometry.
• Dissociation constants (KD) for C5Mab-4 and C5Mab-8 were determined as 3.5 × 10^-8 M and 7.3 × 10^-9 M, respectively.
• C5Mab-4 and C5Mab-8 effectively detected mCCR5 by western blotting.

Conclusions:

• C5Mab-4 and C5Mab-8 are valuable reagents for detecting mouse CCR5 using flow cytometry and western blotting.
• These novel mAbs show potential for use in preclinical studies involving mCCR5.
• The developed mAbs provide a foundation for further investigation into CCR5 function and therapeutic strategies.