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目的 寻找能够特异性检测尿液中3-甲氧基酪胺的单克隆抗体，以满足3-甲氧基酪胺诊断试剂盒开发需要。方法 设计并合成3-甲氧基酪胺抗原，通过3-甲氧基酪胺偶联双琥珀酰亚胺辛二酸酯钠盐和钥孔血蓝蛋白免疫新西兰大白兔获取抗体库，基于噬菌体展示技术，通过固相淘选策略获得高亲和力噬菌体抗体，同时依靠标记生物素抗体包被磁微粒，结合辣根过氧化物酶标记抗原，验证抗体性能。结果 获得20株与3-甲氧基酪胺有较高亲和性的噬菌体抗体，经酶联免疫吸附试验效价测定从中筛选出效价和斜率最优的重组抗体4354#，并以重组抗体4354#偶联生物素为包被抗体，辣根过氧化物酶标记的经酰化3-甲氧基酪胺作为酶标抗原，通过磁微粒化学发光法验证重组抗体4354#可用于检测经酰化处理尿液中3-甲氧基酪胺的含量。测试结果与高效液相色谱法检测结果呈现正相关(R2=0.9021)。结论 通过噬菌体展示技术，成功筛选出针对3-甲氧基酪胺的重组抗体，并通过磁微粒化学发光法验证其可用于检测尿液中3-甲氧基酪胺的含量，为3-甲氧基酪胺诊断试剂盒原材料的开发奠定了基础。
Abstract：Objective To identify a monoclonal antibody capable of specifically detecting urinary 3-methoxytyramine(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 solidphase 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.
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基本信息:
DOI：10.13431/j.cnki.immunol.j.20260016
中图分类号:R392
引用信息:
[1]张航,李娅娅,张伟涛,等.3-甲氧基酪胺噬菌体抗体的制备及应用[J].免疫学杂志,2026,42(02):109-116.DOI:10.13431/j.cnki.immunol.j.20260016.
基金信息:
2024年郑州市优秀青年科技人才（博士）培养专项（郑人社规[2024]5号）; 依托生物医药特色行业学院培养“卓越工程师”的探索与实践（2024SJGLX0627）
2026-02-28
2026-02-28