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

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.

Keywords: Aristolochia; Asarum; Eastern blotting; aristolochic acid; aristololactam; immunostaining; monoclonal antibody.