A sensitive enzyme-linked immunosorbent assay for detecting carcinogenic aristolochic acid in herbal remedies

Aristolochic acid, a naturally occurring nephrotoxin and rodent carcinogen, has been associated with the development of various nephropathies in humans. Developing a sensitive and rapid method to screen the aristolochic acid levels in herbal remedies is urgent for protecting public health. Polyclonal antibodies for aristolochic acid were generated from rabbits after the animals had been immunized with either aristolochic acid-ovalbumin (OVA) or aristolochic acid-keyhole limpet hemocyanin (KLH). A competitive indirect enzyme-linked immunosorbent assay (ciELISA) and a competitive direct enzyme-linked immunosorbent assay (cdELISA) were used for the characterization of the antibodies and for analysis of aristolochic acid contaminated in herbal medicine and diet pills. The antibody titers in the serum of rabbits immunized with aristolochic acid-OVA were considerably higher than those from aristolochic acid-KLH-immunized rabbits. The antibodies from the aristolochic acid-OVA-immunized rabbits were further characterized. In the ciELISA with aristolochic acid-KLH as the plate-coating antigen, the concentrations of the aristolochic acid mixture, aristolochic acid I, and aristolochic acid II that caused 50% inhibition (IC50) of binding of antibodies to aristolochic acid-KLH were found to be 1.2, 0.7, and 18 ng/mL, respectively. When 0.25-5 μg/g of standard aristolochic acid was spiked to ground lotus seeds and then extracted with 0.01 M phosphate-buffered saline, the recovery rate was found to be 86.5% in the ciELISA. Analysis of aristolochic acid in herbal medicine and diet pills with ciELISA showed that 10 of the 12 examined samples were contaminated at levels from 0.6 to 655 μg/g. The presence of aristolochic acid was also confirmed by the high-performance liquid chromatography method.