Preparation and application of 3-methoxytryptamine phage antibody

ZHANG Hang; LI Yaya; ZHANG Weitao; LI Songrui; LIU Yalin; TIAN Xiaoping; LI Guilin; JI Guojie; HU Huanhuan; ZHAO Qiaohui

doi:10.13431/j.cnki.immunol.j.20260016

Abstract：Objective To identify a monoclonal antibody capable of specifically detecting urinary 3-methoxy tyramine(3-MT),so as to underpin the development of a high-performance diagnostic reagent kit.Methods The 3-MT antigen was designed and synthesized.Immune New Zealand white rabbits with 3-MT coupled to bis-succinimidyl suberate sodium salt(BS3)and Keyhole limpet hemocyanin(KLH)to obtain an antibody library.High-affinity phage 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.Results In total,20 phage antibodies with high affinity for 3-MT were obtained.After enzyme-linked immunosorbent assay(ELISA)for titer determination,recombinant antibody 4354# with the optimal titer and slope was selected.Biotin-conjugated recombinant antibody 4354# was used as the coating antibody,and horseradish peroxidase-labeled acylated 3-MT served as the enzyme-labeled antigen.The magnetic microparticle chemiluminescence method verified that recombinant antibody 4354#could be used for competitive detection of 3-MT in acylated urine.The test results showed a significant positive correlation with the HPLC assay results(R2=0.902 1).Conclusion A recombinant antibody targeting 3-MT was successfully screened using phage display technology.its applicability for detecting 3-MT levels in urine was then validated via a magnetic microparticle chemiluminescence method,thereby laying the foundation for the development of raw materials for 3-MT diagnostic kits.