A sensitive and specific indirect competitive enzyme-linked immunosorbent assay for the detection of icariin

Abstract

In the present study, a novel monoclonal antibody (MAb) specific for icariin (ICA) was prepared and characterized. A hybridoma-secreting MAb against icariin was produced by fusing splenocytes immunized with an ICA-bovine serum albumin conjugate with a hypoxanthine-aminopterin-thymidine-sensitive mouse myeloma SP2/0 cell line. The antibody showed high specificity for ICA with almost no cross-reactivity against the majority of structurally-related chemicals. Subsequently, an indirect competitive enzyme-linked immunosorbent assay (ELISA) for ICA was established and characterized. In this assay, an effective measuring range of 10-1,000 ng/ml of ICA (R2=0.9828) was detected. Intra-and inter-assay repeatability and precision were achieved with a relative standard deviation (RSD) of <10%. A mean recovery of 95-115% was obtained, with an RSD of <10%. In addition, the levels of ICA in traditional Chinese herbal prescriptions were determined, and correlation between the ELISA and high-performance liquid chromatography analyses of total ICA was obtained. These results demonstrated that a reliable ELISA method had been successfully developed to determine ICA in traditional Chinese herbs and may contribute to further clinical investigations.