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.
Phytochem Anal
; 24(6): 645-53, 2013.
Article
en En
| MEDLINE
| ID: mdl-23761269
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.Palabras clave
Texto completo:
1
Bases de datos:
MEDLINE
Métodos Terapéuticos y Terapias MTCI:
Terapias_biologicas
Asunto principal:
Plantas Medicinales
/
Immunoblotting
/
Aristolochia
/
Asarum
/
Ácidos Aristolóquicos
Tipo de estudio:
Diagnostic_studies
Idioma:
En
Revista:
Phytochem Anal
Año:
2013
Tipo del documento:
Article
País de afiliación:
Japón