Enzyme-linked immunosorbent assay for glycyrrhizin using anti-glycyrrhizin monoclonal antibody and an eastern blotting technique for glucuronides of glycyrrhetic acid.

Hybridomas secreting a monoclonal antibody against glycyrrhizin were produced by fusing splenocytes from a mouse immunized against a glycyrrhizin-bovine serum albumin conjugate with the hypoxanthine-aminopterin-thymidine-sensitive mouse myeloma cell line, P3-X63-Ag8-653. A very weak cross-reaction with glycyrrhetinic acid monoglucuronide and glycyrrhetic acid occurred. An enzyme-linked immunosorbent assay (ELISA) that had an effective measuring range of 20 -200 ng/mL of glycyrrhizin was established using this monoclonal antibody. In addition, a method named eastern blotting for the detection of glycyrrhizin was investigated. In this method, we developed a new way to separate the glycyrrhizin molecule into two functional parts using a simple and well-known chemical reaction. The sugar parts were oxidized by sodium periodate to give dialdehydes, which reacted with amino groups on the protein and covalently bound to the adsorbent membrane. The monoclonal antibody bound to the aglycone part of the glycyrrhizin molecule for immunostaining. This method was validated by immunocytolocalization of glycyrrhizin in fresh Glycyrrhiza root.