目的 首次合成并鉴定马钱苷(loganin)人工抗原,为进一步制备马钱苷单克隆抗体,建立相关免疫分析方法奠定基础。方法 采用高碘酸钠氧化法合成马钱苷免疫原(loganin-BSA)和包被原(loganin-OVA);应用基质辅助激光解析电离-飞行时间质谱(MALDI-TOF-MS)法鉴定马钱苷人工抗原是否偶联成功;通过酶联免疫吸附试验(enzyme-linkedimmunosorbent assay,ELISA)方法检测免疫小鼠血清中抗马钱苷抗体效价及其特异性。结果 经MALDI-TOF-MS检测,马钱苷与BSA偶联成功。竞争抑制曲线表明,血清中的抗体可与马钱苷产生特异性结合,血清抗体效价在1:40000。结论 成功合成了马钱苷人工抗原,为马钱苷单克隆抗体的制备及其在中药质量评价和动物体内药动学方面的应用奠定了基础。

Objective To synthesize and identify the artificial antigen of loganin for the first time, and provide a foundation for the preparation of specific monoclonal antibody and establishment of immunoassay method. Methods Sodium periodate oxidation method was used to synthesize immunogenic antigen (loganin-BSA) and coating antigen (loganin-OVA) of loganin. Whether loganin was conjugated with BSA and OVA or not was confirmed by matrix-assisted laser desorption ionization/time-of-flight mass spectrometry (MALDI-TOF-MS). The titer and specificity of the antibody in serum of immunised mice were detected by enzyme-linked immunosorbent assay (ELISA). Results The results of MALDI-TOF-MS indicated that loganin was conjugated to BSA. The antibody against loganin obtained from immunised-mice could bind to loganin and the titer was up to 1:40 000. Conclusion The artificial antigen of loganin was synthesized, which can be used further in the preparation of monoclonal antibody, application in quality control of Chinese materia medica and the pharmacokinetic study of loganin in laboratory animals.

国家自然科学基金资助项目(81473590,81473119,81274115);教育部新世纪优秀人才项目(NCET-11-0606);北京市优秀人才培养资助项目(2013D009999000001)