We have been looking for a faster and simpler method for traditional Chinese medicine and natural product assay. In this study, we developed a fluorescent immunoassay approach to detect icariin (ICA) using a fluorescently labelled monoclonal antibody. The ICA-specific antibody was purified by the caprylic acid-ammonium sulphate method and then labelled with rhodamine B isothiocyanate (RBITC). Subsequently, an indirect competitive fluorescence-linked immunosorbent assay (icFLISA) was developed to detect ICA using RBITC-labelled anti-ICA MAbs. The RBITC-labelled monoclonal antibody was highly specific for ICA. The fluorescence assay demonstrated an effective ICA measurement range of 1.28 ng/mL to 20 μg/mL (R2 = 0.9946) with relative standard deviations below 10% for both intra-assay and inter-assay repeatability and precision. This icFLISA for ICA is simple, rapid, and sensitive, with a 20-fold greater linear range and a 10-fold lower limit of detection than with the previously developed indirect competitive enzyme-linked immunosorbent assay (ELISA). Thus, this study establishes a useful method for detecting ICA, enabling in vivo visualization research. In the future, FLISA can be also used to assay the concentrations of ICA in biological samples, as well as to investigate the pharmacokinetics of ICA in different tissues to explore the targets of ICA in vivo.
Keywords: Enzyme-linked immunosorbent assay; Fluorescence-linked immunosorbent assay; Fluorescently labelled monoclonal antibody; Icariin; Rhodamine B isothiocyanate.