中药致肾毒性成分马兜铃酸A单抗制备及酶联免疫分析方法的建立  

Development of Sensitive Monoclonal Antibody-based Enzyme-linked Immunosorbent Assay for Renal Toxicity Ingredient Aristolochic acid A in Chinese Herb Medicine

作　　者：南铁贵[1] 何素平[1] 谭桂玉[1] 李刚[2] 王保民[1] 黄璐琦[3] 

机构地区：[1]中国农业大学农学与生物技术学院,北京100193 [2]吉林省农业科学院农业环境与资源研究中心,长春130033 [3]中国中医科学院中药研究所,北京100700

出　　处：《分析化学》2010年第8期1206-1210,共5页Chinese Journal of Analytical Chemistry

摘　　要：采用活化酯法,将马兜铃酸A分别与牛血清蛋白(BSA)和卵清蛋白(OVA)偶联,得到免疫抗原马兜铃酸A-BSA和包被抗原马兜铃酸A-OVA。利用马兜铃酸A-BSA免疫Balb/c小鼠,制得鼠单克隆抗体1A11,单抗效价为2×104;单抗为IgG1类,轻链为κ型;与其结构类似物马兜铃酸B、C和D的交叉反应率分别为2.8%,3.5%和31.2%。基于抗马兜铃酸A单克隆抗体的间接竞争酶联免疫分析方法(icELISA)的IC50为1.9μg/L,检测范围为0.5～7.5μg/L。icELISA添加回收率为86%～97%,相对标准偏差在5.2%～11.1%之间。利用所建立的icELISA测定了6个中药材和5个中成药中马兜铃酸A的含量,并用高效液相色谱法(HPLC)进行了验证,其中关木通、广防己、天仙藤、马兜铃和青木香中均检测出马兜铃酸A,而川木通和5个中成药中未检测到马兜铃酸A。结果表明:本方法可用于中药中马兜铃酸A的快速检测。Aristolochic acid A-bovine serum albumin （BSA） conjugate and aristolochic acid A-ovalbumin （OVA） conjugate,were produced as the immunogen and coating antigen,respectively. A monoclonal antibody （mAb）,designated as 1A11,was produced with aristolochic acid A-BSA. The titer was 2×10^4. The mAb was IgG1 isotype with κ light chains. A sensitive monoclonal antibody-based indirected enzyme-linked immunosorbent assay （icELISA） was established with the mAb. The cross-reactivity （CR） of mAb 1A11 with aristolochic acid B,C and D,an analog of aristolochic acid A,was 2.8%,3.5% and 31.2%,respectively. IC50 and the working range of the icELISA for aristolochic acid A were 1.9 and 0.5-7.5 μg/L,respectively. The average recoveries of aristolochic acid A fortified in caulis clematidis armandii were 86%-97% with RSD of 5.2%-11.1%. Analysis of aristolochic acid A in Chinese herb medicines and Chinese proprietary medicines with icELISA showed that the examined samples of manshurian dutchmanspipe stem,herba aristolochiae,fructus aristolochiae,aristolochia fangchi,and dutchmanspipe root were positive,the caulis clematidis armandii and the other Chinese proprietary medicines examined samples were negative and the results were confirmed by HPLC analysis. This ELISA is suitable for the rapid detection of aristolochic acid A in Chinese Herb medicine.

关 键 词：马兜铃酸A 单克隆抗体 中药 酶联免疫分析法 

分 类 号：R285[医药卫生—中药学]