Abstract
In this study, a specific monoclonal antibody (mAb) against Alternaria mycotoxin tenuazonic acid (TeA) was prepared and a sensitive indirect competitive enzyme-linked immunosorbent assay (ic-ELISA) for the detection of TeA was developed. Tenuazonic acid coupled with carboxymethoxylamine hemihydrochloride (TeA-CMO) was conjugated to keyhole limpet hemocyanin (KLH) as an immunogen for Balb/c mice injection. The hybridoma cell line 3F10 secreting the mAb specific to TeA was obtained, and then by intraperitoneal injection and the caprylic acid-ammonium sulfate precipitation, the purified mAb was prepared and identified as the immunoglobulin G1 isotypes. The affinity constant (kD) of the mAb was 3.045 E−3 M by SPR analysis. Based on this mAb, an ic-ELISA was established after optimization of assay condition, namely the concentration of coating antigen and antibody were 250 ng/mL and 125 ng/mL respectively, competition time of antigen-antibody was 40 min, and incubation time of secondary antibody was 40 min in 0.01 M PBS buffer (pH 7.4). Under the optimized condition, the IC50 value and the detection limit (LOD) were 18.50 ng/mL and 1.00 ng/mL respectively. The average recovery rate from spiked beer, apple juice, and grape juice was from 85.0 to 120.0%. A squared coefficient of correlation (R2) between ic-ELISA and HPLC method was 0.9732. The established ic-ELISA provides an acceptable technique for the detection of TeA residue in food samples.
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Funding
This research was funded by the Key Area R&D Program of Guangdong Province (No. 2019B020211002), the National Natural Science Foundation of China (No. 31972157), the Science and Technology Planning Project of Guangzhou City (201804020077), and the International Cooperation Program of SCAU (2019SCAUGH03) and Project Supported by Guangdong Province Universities and Colleges Pearl River Scholar Funded Scheme (2017).
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Yi-Fan Liang declares that he has no conflict of interest. Xiao-Wen Zhou declares that he has no conflict of interest. Feng Wang declares that he has no conflict of interest. Yu-Dong Shen declares that he has no conflict of interest. Zhi-Li Xiao declares that she has no conflict of interest. Shi-Wei Zhang declares that he has no conflict of interest. Yong-Jun Li declares that he has no conflict of interest. Hong Wang declares that she has no conflict of interest.
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Female Balb/c mice were housed and maintained at the Guangdong Medical Laboratory Animal Center. All animal experiments were performed in compliance with the protective and administrative laws for laboratory animals of China and conducted with the approval of the Institutional Authority for Laboratory Animal Care, South China Agricultural University, Guangzhou, China.
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Liang, YF., Zhou, XW., Wang, F. et al. Development of a Monoclonal Antibody-Based ELISA for the Detection of Alternaria Mycotoxin Tenuazonic Acid in Food Samples. Food Anal. Methods 13, 1594–1602 (2020). https://doi.org/10.1007/s12161-020-01780-w
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DOI: https://doi.org/10.1007/s12161-020-01780-w