Preparation of Anti-Glycyrrhetinic Acid Monoclonal Antibody for Application in an Indirect Competitive Enzyme-Linked Immunosorbent Assay

摘要

Glycyrrhetinic acid is a major metabolite of glycyrrhizin, which is one of the main components of licorice roots and is considered to be one of the pharmacologically active substances in licorice. A new hybridoma cell line, named G-2A6, was generated by fusing mouse myeloma cells and splenocytes, which were immunized using glycyrrhetinic-acid-keyhole limpet hemocyanin to produce a monoclonal antibody (mAb) against glycyrrhetinic acid. Using the anti-glycyrrhetinic acid mAb, we attempted to develop a simple, rapid, and highly sensitive indirect competitive enzyme-linked immunosorbent assay (icELISA). The developed icELISA had a range from 3.91 to 125ng/mL with low coefficients of variation (less than 5%) and demonstrated a high recovery rate of glycyrrhetinic acid spiked into licorice powder (average=101.76%). In addition, the icELISA could determine the glycyrrhetinic acid concentration in glycyrrhetinic-acid-spiked human serum with simple pretreatment, which suggests that the developed ELISA system using anti-glycyrrhetinic acid mAb would prove to be an effective and useful tool for determining glycyrrhetinic acid in various fields such as the analysis of Glycyrrhiza plants and pharmacokinetic studies of glycyrrhetinic acid during the administration of glycyrrhetinic acid, glycyrrhizin, and/or licorice-based medical agents.