Sandwich Enzyme-linked Immunosorbent Assay (ELISA) to Quantify Monoclonal Antibody (B[a]P-13) for Herbal Medicine Products

Abstract

Abstract: Sandwich enzyme-linked immunosorbent assay (ELISA) to quantify monoclonal antibody (B[a]P-13) Background: Only a few studies have focused on the analysis using specific antibodies in the sandwich ELISA method to each B[a]P in herbal medicine products. In contrast to the sandwich ELISA method, many competitive ELISA methods using specific antibodies such as benzo[a]pyrene monoclonal antibody (B[a]P-13) and a goat anti-mouse IgG (H+L) cross-adsorbed secondary antibody, horseradish peroxidase (HRP) were developed. Introduction: The objective of this study was to develop and validate the method for the response of the benzo[a]pyrene monoclonal antibody (B[a]P-13) and goat anti-mouse IgG (H+L) cross-adsorbed secondary antibody (HRP) to prepare the immunogen and its application to detect the benzo[a]pyrene in various herbal medicine products. Methods: This research method includes preparation of B[a]P-protein conjugates, sampling and extraction procedure for herbal medicines, sandwich ELISA procedure, evaluation of cross-reactivity for determination, matrix effect of the organic solvents, correlation of benzo[a]pyrene detection ELISA compared to HPLC-FLD in herbal medicine products. Results: The sandwich ELISA method for B[a]P was validated in linearity (R2 > 0.99), the limit of detection (LOD) (0.080.19 μg/kg) and limit of quantification (LOQ) (0.240.57 μg/kg), accuracy (95.58117.06 %), and precision (3.8010.26 %). The cross-reactivity (CR) was found for B[a]P (100%), CHR (39%), B[b]F (27%), and B[a]A (41%). As a solvent, acetonitrile (MeCN) was used to express the normalized sandwich ELISA calibration curves with benzo[a]pyrene monoclonal antibody (B[a]P-13). The antigen-antibody binding in sandwich ELISA was decreased about 10 times with increasing the salt content (0.0060.18 mol/L phosphate to 20400 mmol/L). The pH range from 6 to 9 was not considered to affect the performance of the sandwich ELISA. Correlation of B[a]P detection in herbal medicines with ELISA compared to HPLC-FLD expressed good correlation (R2 = 0.991) and the slope of the graph for the ELISA (B[a]P-equivalents μg/kg) value divided by the HPLC-FLD (B[a]P μg/kg) value was 0.7292. Conclusion: Therefore, sandwich ELISA method using benzo[a]pyrene monoclonal antibody (B[a]P-13) could be an alternative screening method for detection of B[a]P in herbal medicine products.