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The Natural Products Journal


ISSN (Print): 2210-3155
ISSN (Online): 2210-3163

Research Article

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

Author(s): Yong-Yeon Kim and Han-Seung Shin*

Volume 13, Issue 3, 2023

Published on: 13 December, 2022

Article ID: e041122210658 Pages: 9

DOI: 10.2174/2210315513666221104154116

Price: $65


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.

Keywords: Herbal medicine products, benzo[a]pyrene monoclonal antibody (B[a]P-13), goat anti-mouse IgG (H+L) crossadsorbed secondary antibody horseradish peroxidase (HRP), sandwich enzyme-linked immunosorbent assay, cross-reactivity, solvent, acetonitrile.

Graphical Abstract
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