title: 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.
abstract: BACKGROUND
  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.
  
  
  CONCLUSIONS
  This method was extremely useful for the visual screening of AA-I and AA-II among easily mistaken herbal medicines.
authors.affiliation: 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.
authors.forename: Xiao-Wei
authors.initials: XW
authors.lastname: Li
authors.forename: Osamu
authors.initials: O
authors.lastname: Morinaga
authors.forename: Min
authors.initials: M
authors.lastname: Tian
authors.forename: Takuhiro
authors.initials: T
authors.lastname: Uto
authors.forename: Jie
authors.initials: J
authors.lastname: Yu
authors.forename: Ming-Ying
authors.initials: MY
authors.lastname: Shang
authors.forename: Xuan
authors.initials: X
authors.lastname: Wang
authors.forename: Shao-Qing
authors.initials: SQ
authors.lastname: Cai
authors.forename: Yukihiro
authors.initials: Y
authors.lastname: Shoyama
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal; D030022:Aristolochia / Q000737:chemistry*; D034341:Aristolochic Acids / Q000032:analysis*; D030023:Asarum / Q000737:chemistry*; D015151:Immunoblotting / Q000379:methods*; D008297:Male; D051379:Mice; D008807:Mice, Inbred BALB C; D010946:Plants, Medicinal / Q000737:chemistry*
chemical_list: D000911:Antibodies, Monoclonal; D034341:Aristolochic Acids; C000228:aristolochic acid I; C042310:aristolochic acid II
keywords: Aristolochia; Asarum; Eastern blotting; aristolochic acid; aristololactam; immunostaining; monoclonal antibody