Development of an Eastern Blotting Technique for the Visual Detection of Aristolochic Acids in Aristolochia and Asarum Species by Using a Monoclonal Antibody Against Aristolochic Acids I and II
Development of an Eastern Blotting Technique for the Visual Detection of Aristolochic Acids in Aristolochia and Asarum Species by Using a Monoclonal Antibody Against Aristolochic Acids I and II
复制标题
使用抗马兜铃酸 I 和 II 的单克隆抗体开发东部印迹技术，用于视觉检测马兜铃和细辛中的马兜铃酸
DOI：
10.1002/pca.2448
复制
发表时间：
2013-11-01
影响因子：
3.3
通讯作者：
Shoyama, Yukihiro
中科院分区：
文献类型：
--
作者：
Li, Xiao-Wei;Morinaga, Osamu;Shoyama, Yukihiro
马兜铃酸（Aristolochic Acids，AAs）是一种天然存在的肾毒性物质和人类致癌物。马兜铃酸Ⅰ（AA-Ⅰ）和马兜铃酸Ⅱ（AA-Ⅱ）是两种毒性明显的重要氨基酸，目的获得识别AA-Ⅰ和AA-Ⅱ的单克隆抗体（MAb），并建立特异性显示和简便检测AA-Ⅰ和AA-Ⅱ的Eastern印迹技术。方法采用脾细胞融合法制备分泌抗AAs单克隆抗体的杂交瘤细胞株来源于用AA-I-钥孔血蓝蛋白（KLH）缀合物和骨髓瘤细胞系SP2/0-Ag 14免疫的小鼠。AA-I和AA-II通过薄层色谱法（TLC）分离，然后使用改进的碳二亚胺法印迹到带正电荷的聚醚砜（PES）膜上。将所得膜结合AA-蛋白缀合物连接到新制备的MAb，然后连接到用过氧化物酶标记的二抗。结果MAb 2A 10 - 10 B对AA-Ⅰ（100%）和AA-Ⅱ（69.3%）具有较高的特异性，对可能干扰植物中AA-Ⅰ和AA-Ⅱ检测的类似物具有较低的交叉反应性（2.2%）。一个建立东部印迹法应用于AA-I和AA-II在干燥的植物组织中的免疫组化定位，这一分析表明，Aristolochia manshuriensis干的外胚层，皮层和韧皮部可能含有较高的总AA-I和AA-II的量相比，髓和木质部。版权所有（c）2013 John Wiley & Sons，Ltd.获得了同时识别马兜铃酸I和II的灵敏单克隆抗体（MAb）。作为单克隆抗体2A 10 - 10 B的应用之一，我们建立了一种新的Western印迹法，用于马兜铃属和细辛属植物中AA-I和AA-II的目视筛选。所建立的Eastern blotting方法也适用于AA-Ⅰ和AA-Ⅱ在干植物组织中的免疫组织定位。
IntroductionAristolochic acids (AAs) are naturally occurring nephrotoxicants and human carcinogens. Aristolochic acid I (AA-I) and aristolochic acid II (AA-II) are two important AAs with clear toxicity.ObjectiveTo obtain a monoclonal antibody (MAb) recognising AA-I and AA-II and develop an Eastern blotting technique for the specific visualisation and easy determination of AA-I and AA-II in plant extracts or tissues of Aristolochia and Asarum species.MethodsA hybridoma secreting MAb against AAs was prepared by cell fusion with splenocytes derived from a mouse immunised with AA-I-keyhole limpet haemocyanin (KLH) conjugate and the myeloma cell line SP2/0-Ag14. AA-I and AA-II were separated by thin-layer chromatography (TLC) and then blotted onto a positively charged polyethersulphone (PES) membrane using a modified carbodiimide method. The resulting membrane-bound AA-protein conjugates were linked to the newly prepared MAb and then to the secondary antibody labelled with peroxidase. 4-Chloro-1-naphthol was then added as the peroxidase substrate for staining.ResultsMAb 2A10-10B showed a high specificity for AA-I (100%) and AA-II (69.3%) and low cross reactivity ( 2.2%) toward analogues that may disrupt detection of AA-I and AA-II in plants. An established Eastern blotting method was applied to the immunohistolocalisation of AA-I and AA-II in dry plant tissues, and this analysis showed that the phelloderm, cortex and phloem of Aristolochia manshuriensis stem may contain higher amounts of total AA-I and AA-II as compared with the pith and xylem.ConclusionThis method was extremely useful for the visual screening of AA-I and AA-II among easily mistaken herbal medicines. Copyright (c) 2013 John Wiley & Sons, Ltd.A sensitive monoclonal antibody (MAb) which simultaneously recognises aristolochic acids I and II was obtained. As one of the application of MAb 2A10-10B, an Eastern blotting method was newly developed for the visual-screening of AA-I and AA-II in Aristolochia and Asarum species. The established Eastern blotting method was also applied to the immunohistolocalisation of AA-I and AA-II in dry plant tissues.