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| Establishment of an indirect competitive ELISA detection method for fluoroquinolones in livestock farm wastewater |
| Received:April 29, 2025 |
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| KeyWord:fluoroquinolones;monoclonal antibodies against norfloxacin;indirect competitive enzyme-linked immunosorbent assay（ic-ELISA）;swine farm wastewater |
| Author Name | Affiliation | E-mail | | CHI Jiayao | College of Veterinary Medicine, Shanxi Agricultural University, Jinzhong 030801, China
Chinese Center for Animal Health and Epidemiology, Qingdao 266032, China | | | LI Zehui | College of Veterinary Medicine, Shanxi Agricultural University, Jinzhong 030801, China
Chinese Center for Animal Health and Epidemiology, Qingdao 266032, China | | | FANG Hanqing | Chinese Center for Animal Health and Epidemiology, Qingdao 266032, China
Qingdao Key Laboratory of Modern Bioengineering and Animal Disease Research, Qingdao 266032, China | | | LI Muzi | Chinese Center for Animal Health and Epidemiology, Qingdao 266032, China
Qingdao Key Laboratory of Modern Bioengineering and Animal Disease Research, Qingdao 266032, China | | | WANG Tong | Chinese Center for Animal Health and Epidemiology, Qingdao 266032, China
College of Veterinary Medicine, Tianjin Agricultural College, Tianjin 300384, China | | | LI Jiamin | College of Veterinary Medicine, Shanxi Agricultural University, Jinzhong 030801, China
Chinese Center for Animal Health and Epidemiology, Qingdao 266032, China | | | CHANG Weifan | College of Veterinary Medicine, Shanxi Agricultural University, Jinzhong 030801, China
Chinese Center for Animal Health and Epidemiology, Qingdao 266032, China | | | HUO Nairui | College of Veterinary Medicine, Shanxi Agricultural University, Jinzhong 030801, China | tgnrhuo@163.com | | CHANG Hong | College of School of Food Science and Engineering, Shanxi Agricultural University, Jinzhong 030801, China | 941004578@qq.com | | ZHAO Sijun | Chinese Center for Animal Health and Epidemiology, Qingdao 266032, China
Qingdao Key Laboratory of Modern Bioengineering and Animal Disease Research, Qingdao 266032, China | zhsjun@126.com |
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| Abstract: |
| To establish an indirect competitive enzyme-linked immunosorbent assay（ic-ELISA） for the detection of fluoroquinolones（FQs） in wastewater, providing technical support for the regulation of drug use in animal husbandry and environmental monitoring. Monoclonal antibodies were prepared by immunizing mice with bovine serum albumin-norfloxacin（NOR）. The FQs-specificity of NOR antibodies and their cross-reactivity with eight other FQs were examined. The optimal coating concentration of OVA-NOR, the optimal dilution of monoclonal antibodies, and the optimal dilution of enzyme-labeled secondary antibodies were determined using a matrix method. The matrix effect and detection sensitivity of the ic-ELISA were evaluated using FQs-negative wastewater samples spiked with standard substances. Four cell lines that stably secreted NOR monoclonal antibodies were selected. The secreted antibodies were all of the IgG1 subclass and Kappa type. These antibodies showed no reactivity with sulfonamides, tetracyclines, or macrolides. Among them, the monoclonal antibody secreted by the 2F11 cell line exhibited cross-reactivity rates of 3.93% to 139.14% with nine FQs and had the lowest IC50 values and the highest reactivity rates for three discontinued drugs. Based on the 2F11 cell line monoclonal antibody, the ic-ELISA detection method was established. It was determined that wastewater samples should be processed by centrifugation to remove impurities, followed by microfiltration and a 5-fold dilution. The optimal coating concentration of OVA-NOR was 0.64 μg·mL-1, the addition concentration of NOR monoclonal antibody was 1.20 μg·L-1, and the enzyme-labeled secondary antibody was diluted at 1∶20 000. The spiking experiments showed that the nine FQs had good linear relationships in the range of 0.3-20 μg·L-1, with R2 values all greater than 0.997. The detection limit of the method was 0.9 μg·L-1, and the quantification limit was 3 μg·L-1. The average recovery rates of the nine drugs in the spiking recovery experiments（60.6%-107.1%） were higher than those of the UPLC-MS/MS method（22.0%-73.6%）. The intra- and inter-batch RSD values were 1.7%-11.2% and 4.1%-12.9%, respectively, both lower than those of the UPLC-MS/MS method. The ic-ELISA detection method based on NOR monoclonal antibodies is suitable for the sensitive detection of nine FQs in aquaculture wastewater. It requires less stringent sample pretreatment and is more economical and convenient. |
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