FIGURE 4.

Development of enzyme-linked immunosorbent assay (ELISA) system to measure concentrations of 3 in human serum samples using anti-18β-glycyrrhetyl-3-O-glucuronide (3MGA)-monoclonal antibody (mAb). (A) Dot-blot analysis of glycyrrhizin (GL) metabolites using anti-3MGA-mAb. GL metabolites and bovine serum albumin (BSA, 1 µg each) were spotted onto polyethersulfone membrane and stained using an anti-3MGA-mAb. (B) Competitive ELISA using an anti-3MGA-mAb for 3MGA, 3, and 4 in aqueous solution was performed (C) Standard solutions of 3 (15.6 nM–2 µM) in normal human serum were prepared. Serum samples were deproteinized by treating with 80% ethanol, followed by centrifugation, and the supernatants were dried up under reduced pressure. Standard lines between absorbance and concentrations of compound 3 are shown (D) Accuracy of ELISA system in measuring concentrations of 3 in human serum samples using anti-3MGA-mAb. Human serum samples were deproteinized, and concentration of 3 was measured using ELISA system. Samples with observed values higher than 400 nM were diluted appropriately with H₂O to set detectable range from 10 to 400 nM. Values were compared with the result of liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis from our previous study (Takahashi K, 2019). Significant relationship was observed between the values observed using ELISA and LC-MS/MS values (r ² = 0.91).