State Key Laboratory of Natural and Biomimetic Drugs, School of Pharmaceutical Sciences, Peking University, Beijing, 100191, PR China; Department of Pharmacognosy, Faculty of Pharmaceutical Sciences, Nagasaki International University, Sasebo, Nagasaki, 859-3298, Japan.
Phytochem Anal. 2013 Nov-Dec;24(6):645-53. doi: 10.1002/pca.2448. Epub 2013 Jun 13.
Aristolochic 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.
To 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.
A 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.
MAb 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.
This method was extremely useful for the visual screening of AA-I and AA-II among easily mistaken herbal medicines.
马兜铃酸（AAs）是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）是两种具有明确毒性的重要 AAs。
获得识别 AA-I 和 AA-II 的单克隆抗体（MAb），并开发一种 Eastern 印迹技术，用于特异性可视化和容易确定马兜铃属和细辛属植物提取物或组织中的 AA-I 和 AA-II。
通过细胞融合，用 AA-I-钥孔血蓝蛋白（KLH）缀合物免疫的小鼠的脾细胞与骨髓瘤细胞系 SP2/0-Ag14 制备分泌针对 AAs 的杂交瘤。用薄层色谱法（TLC）分离 AA-I 和 AA-II，然后用改良的碳二亚胺法将其印迹到带正电荷的聚醚砜（PES）膜上。所得膜结合的 AA-蛋白缀合物与新制备的 MAb 结合，然后与标记过氧化物酶的二级抗体结合。然后将 4-氯-1-萘酚作为过氧化物酶的底物进行染色。
MAb 2A10-10B 对 AA-I（100%）和 AA-II（69.3%）具有高度特异性，对可能干扰植物中 AA-I 和 AA-II 检测的类似物的交叉反应性≤2.2%。建立的 Eastern 印迹法被应用于干植物组织中 AA-I 和 AA-II 的免疫组织化学定位，该分析表明马兜铃属茎的韧皮部、皮层和韧皮部可能含有比髓和木质部更高量的总 AA-I 和 AA-II。
该方法对于容易混淆的草药中 AA-I 和 AA-II 的视觉筛选非常有用。
