DEVELOPMENT AND APPLICATION OF A SANDWICH ENZYME-IMMUNOASSAY FOR GLYCYRRHIZAE RADIX PROTEIN (GRP) USING MONOCLONAL-ANTIBODIES

We hare developed mouse monoclonal antibodies (anti-GRP mAb-1-5, all I gG1 sub-isotype mAbs) against Glycyrrhizae Radix protein (GRP), which was recently determined to be a marker protein of Glycyrrhizae Radix ( GR). Among these, anti-GRP mAb-1 and 2 were found to recognize differe nt epitopes on the GRP molecule, as demonstrated by ELISA analysis, an d were used for the development of a sandwich enzyme immunoassay (SEIA ) for GRP in traditional Chinese medicines (TCMs), The SEIA was based on the principle of binding an analyte to anti-GRP mAb2 coated on poly styrene microtiter wells, followed bg immunoreaction with biotinylated anti-GRP mAb1 and horseradish peroxidase-streptavidin. The SEIA was s pecific to GRP in GRs, and showed no cross-reaction with any Leguminos ae crude: drugs other than GRs. This SEIA detected CRP with excellent reproducibility (coefficient of variation=5.9%), an EC50 of 11.5 ng/we ll and a detection limit of 0.1 ng/well, The present SEIA was about 10 -times more sensitive in detecting GRP than the selected antibody enzy me immunoassay (SAEIA) for GRP previously developed using an antiserum to GR itself Also, the SEIA has such a low assay background that it a llowed us to detect a low concentration of GRP in Kyuki-tyoketsu-in-da iichi-kagen (KTIDK), a TCM consisting of only 2.7% GR, The GRF SEA was simple, accurate, reproducible and may pro,ide a general analytical m ethod for the quality control of GR-based TCMs.