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

• Published in: Phytochemical analysis 2013-11, Vol.24 (6), p.645-653
• Main Authors: Li, Xiao-Wei, Morinaga, Osamu, Tian, Min, Uto, Takuhiro, Yu, Jie, Shang, Ming-Ying, Wang, Xuan, Cai, Shao-Qing, Shoyama, Yukihiro
• Format: Article
• Language: English
• Subjects: Acids; Animals; Antibodies, Monoclonal; Aristolochia; Aristolochia - chemistry; aristolochic acid; Aristolochic Acids - analysis; aristololactam; Asarum; Asarum - chemistry; Eastern blotting; Immunoblotting - methods; immunostaining; Male; Mice; Mice, Inbred BALB C; monoclonal antibody; Plants, Medicinal - chemistry
• ISSN: 0958-0344; 1099-1565
• DOI: 10.1002/pca.2448

ABSTRACT Introduction 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. Objective 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. Methods 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. Results 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. Conclusion This method was extremely useful for the visual screening of AA‐I and AA‐II among easily mistaken herbal medicines. Copyright © 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.