Development and application of a sandwich enzyme immunoassay for Glycyrrhizae Radix protein (GRP) using monoclonal antibodies.
We have developed mouse monoclonal antibodies (anti-GRP mAb-1-5, all IgG1 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 different epitopes on the GRP molecule, as demonstrated by ELISA analysis, and 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 polystyrene microtiter wells, followed by immunoreaction with biotinylated anti-GRP mAb1 and horseradish peroxidase-streptavidin. The SEIA was specific to GRP in GRs, and showed no cross-reaction with any Leguminosae crude drugs other than GRs. This SEIA detected GRP with excellent reproducibility (coefficient of variation=5.9%), an EC50 of 11.5 ng/well 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 enzyme immunoassay (SAEIA) for GRP previously developed using an antiserum to GR itself. Also, the SEIA has such a low assay background that it allowed us to detect a low concentration of GRP in Kyuki-tyoketsu-in-daiichi-kagen (KTIDK), a TCM consisting of only 2.7% GR. The GRP SEIA was simple, accurate, reproducible and may provide a general analytical method for the quality control of GR-based TCMs.
Bai
G
G
Faculty of Pharmaceutical Sciences, Nagasaki University, Nagasaki, Japan.
Fujiwara
K
K
Tanimori
H
H
Kitagawa
T
T
D000818:Animals; D000911:Antibodies, Monoclonal; D019921:Biotinylation; D004355:Drug Stability; D004365:Drugs, Chinese Herbal / Q000032:analysis*; D004797:Enzyme-Linked Immunosorbent Assay; D006735:Horseradish Peroxidase; D007124:Immunoenzyme Techniques; D051379:Mice; D008807:Mice, Inbred BALB C; D010940:Plant Proteins / Q000032:analysis* / Q000737:chemistry; D019809:Streptavidin
D000911:Antibodies, Monoclonal; D004365:Drugs, Chinese Herbal; D010940:Plant Proteins; D019809:Streptavidin; D006735:Horseradish Peroxidase