免疫亲和色谱特异性剔除中药方剂四逆散中的柚皮苷  

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

作　　者：陈亮[1] 陈婷[1] 徐强[1] 

机构地区：[1]南京大学生命科学学院医药生物技术国家重点实验室,江苏南京210093

出　　处：《色谱》2006年第3期243-246,共4页Chinese Journal of Chromatography

基　　金：国家自然科学基金重大研究计划资助项目(No.90209040)

摘　　要：为了获得剔除柚皮苷(naring in)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naring in-B SA,并用naring in-B SA对新西兰兔进行免疫获得抗血清,再将其纯化后与经CNB r活化的Sepharo se4B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,naring in-B SA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∶30 000,抗体IgG的纯度达94%,交叉反应率低。在IgG与Sepha-ro se4B合成的IgG-Sepharo se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。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 ： 30 000, 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.

关 键 词：免疫亲和色谱 柚皮苷 完全抗原 特异性剔除 四逆散 

分 类 号：O658[理学—分析化学]