Preparation and Application of Monoclonal Antibody Against 3-Methoxydopamine

Objective This study aims to prepare monoclonal antibodies against 3-methoxydopamine and verify the antibody performance via magnetic microparticle chemiluminescence method.Method The 3-methoxydopamine antigen was designed and synthesized.Immune New Zealand white rabbits with 3-methoxydopamine coupled to bis-succinimidyl suberate sodium salt (BS3) and Keyhole limpet hemocyanin (KLH) to obtain an antibody library.High-affinity monoclonal antibodies were obtained using solid- phase selection strategy based on phage display technology.Antibody performance was verified by coating magnetic particles with biotin-labeled antibodies and combining them with horseradish peroxidase-labeled antigen.Result Through phage display technology, 20 monoclonal antibodies with high affinity for 3-methoxydopamine were obtained. After ELISA titer determination, monoclonal antibody 4354# with the best titer and gradient was selected. Biotin-conjugated monoclonal antibody 4354# was used as the coating antibody, and horseradish peroxidase-labeled 3-methoxydopamine served as the enzyme-labeled antigen. The magnetic microparticle chemiluminescence method verified that monoclonal antibody 4354# can be used for competitive detection of 3-methoxydopamine in urine. The test results showed a significant positive correlation with the HPLC assay results (R² = 0.9021).Conclusion A monoclonal antibody against 3-methoxytyramine was successfully isolated using phage display technology. It was then validated via a magnetic microparticle chemiluminescence method for its ability to quantitatively detect 3-methoxytyramine in urine through a competitive method. This work has laid the foundation for the development of materials for 3-methoxytyramine diagnostic kits.