色谱

色谱

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免疫亲和色谱特异性剔除中药方剂四逆散中的柚皮苷

陈亮 陈婷 徐强   

  1. State Key Laboratory of Pharmaceutical Biotechnology, School of Life Sciences, Nanjing University, Nanjing 210093, China
  • 收稿日期:2005-11-30 修回日期:2006-03-08 出版日期:2006-05-30 发布日期:1987-03-25
  • 通讯作者: 陈婷

Specific Depletion of Naringin from Si-Ni-San, a Traditional Chinese Prescription, by an Immunoaffinity Chromatography

CHEN Liang, CHEN Ting, XU Qiang   

  1. State Key Laboratory of Pharmaceutical Biotechnology, School of Life Sciences, Nanjing University, Nanjing 210093, China
  • Received:2005-11-30 Revised:2006-03-08 Online:2006-05-30 Published:1987-03-25
  • Contact: Chen Ting

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摘要： 为了获得剔除柚皮苷(naringin)的中药方剂四逆散样品以供其药理活性探讨时使用,制备了抗柚皮苷抗体的免疫亲和色谱柱,用于特异性地剔除四逆散中的柚皮苷。首先合成了柚皮苷的完全抗原柚皮苷与牛血清白蛋白的结合物naringin-BSA,并用naringin-BSA对新西兰兔进行免疫获得抗血清,再将其纯化后与经CNBr活化的Sepharose 4B凝胶共价偶联制成免疫亲和色谱柱。将四逆散提取物样品溶液上样该色谱柱,洗脱,制得特异性剔除了柚皮苷的四逆散样品。由检测结果可知，naringin-BSA被成功合成。将其用于免疫新西兰兔,获得的抗血清的效价经酶联免疫吸附法（ELISA）测定达到1∶30000,抗体IgG的纯度达94%,交叉反应率低。在IgG与Sepharose 4B合成的IgG-Sepharose免疫亲和色谱柱中,IgG的偶联率为87%。用该免疫亲和色谱柱处理四逆散后,其中所含的柚皮苷几乎完全被剔除。结果证明,利用抗柚皮苷免疫亲和色谱,能特异性地剔除四逆散或其他样品中的柚皮苷成分。

关键词: CNBr活化的Sepharose 4B , 免疫亲和色谱, 四逆散, 特异性剔除, 完全抗原, 柚皮苷

Abstract: To specifically deplete the compound naringin from Si-Ni-San, a traditional Chinese prescription, an immunoaffinity chromatography column was prepared. The naringin bovine serum albumin (BSA) complex (naringin-BSA) was synthesized to make the complete antigen naringin-BSA. Polyclonal antibody was prepared from the serum of rabbit immunized with naringin-BSA. Then, an immunoaffinity column was made by covalently coupling the polyclonal antibody to CNBr-activated Sepharose 4B and used for depleting naringin from Si-Ni-San. The polyclonal antibody obtained from the rabbit serum was found through enzyme linked immunosorbent assay (ELISA) to show the titer of 1∶30000, the purity of 94% and low cross-reaction rate. The coupling rate of the polyclonal antibody to Sepharose 4B was 87%. By using this column, naringin in Si-Ni-San was selectively depleted from the whole extract. This immunoaffinity column of anti-naringin antibody could be used for specifically depleting naringin from Si-Ni-San and other samples.

Key words: CNBr-activated Sepharose 4B , complete antigen, Immunoaffinity column, Naringin, Si-Ni-San, specific depletion