Abstract
Rhein is an important quality-control marker of Rheum officinale. The aim of this study was to develop an indirect competitive enzyme-linked immunosorbent assay (icELISA) for rhein detection, which acts as a powerful tool for quality control and proper usage of Rheum officinale. First, a specific and sensitive monoclonal antibody (mAb) against rhein was produced from a stable hybridoma cell line, 1F8, generated by the fusion of mouse myeloma sp2/0 with spleen cells obtained from a Bal b/c mouse immunized with rhein-BSA. Then, an icELISA method was developed with an IC50 value and working range of 0.05 μg L−1 and 0.02–0.11 μg L−1, respectively. The icELISA revealed high assay specificity, since it only had a relatively high cross reactivity with aloe-emodin (27%) and almost no cross reactivity with any other anthraquinones (<1%). When spiked with 0.2–2 mg kg−1 of rhein, the recoveries ranged from 84.19% to 102.90%. Finally, icELISA was used to detect rhein contents of Rheum officinale collected from different regions, and the results corresponded well with those of HPLC. Overall, the developed icELISA with high specificity and sensitivity provided a rapid and simple method for rhein detection, and it may be a powerful tool for quality control and proper usage of Rheum officinale.
Highlights
Rheum officinale, one of the oldest and best-known traditional Chinese medicines, has been used as a remedy for constipation and fever for thousands of years and is officially listed in many pharmacopoeias, such as China Pharmacopoeia, British Pharmacopoeia, European Pharmacopoeia, Korean Pharmacopoeia, and Japan Pharmacopoeia [1,2,3,4,5]
Erefore, rhein-BSA was prepared as immunogen, while rhein-OVA was prepared as coating antigen for indirect competitive enzyme-linked immunosorbent assay (icELISA) in
Aside from the mAb1F8 that we developed in this study, Zhang [26] produced an antibody against rhein from eggs of immunized female roman chickens with rhein-BSA. e egg-yolk antibody had a high cross reactivities (CRs) with rheum emodin (64.58%), while the CRs with aloe-emodin, physcion, and chrysophanol were unanalyzed
Summary
One of the oldest and best-known traditional Chinese medicines, has been used as a remedy for constipation and fever for thousands of years and is officially listed in many pharmacopoeias, such as China Pharmacopoeia, British Pharmacopoeia, European Pharmacopoeia, Korean Pharmacopoeia, and Japan Pharmacopoeia [1,2,3,4,5]. Us, establishing an efficient and rapid method for determination of rhein concentrations is important for the quality control and proper usage of Rheum officinale. A variety of analytical methods, including high-performance thin-layer chromatography (HPTLC) [12], high-performance liquid chromatography (HPLC) [13], ultra-performance liquid chromatography (UPLC) [14], capillary electrophoresis (CE) [15,16,17], and electrochemical methods [18], have been used for quantitative detection of rhein in Rheum officinale and other herbs. Immunoassays based on antibody-antigen specific recognition have played an important role as analytical tools for the quality control of traditional Chinese medicines. A specific monoclonal antibody (mAb) against rhein was produced Based on this mAb, an icELISA method for rhein detection was developed and successfully applied for the determination of rhein in different Rheum officinale samples, the results of which were verified by HPLC
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