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Abstract: Fumonisin B1 (FB1) is a mycotoxin, mainly produced by Fusarium fungi and present in food and feed. It causes harmful effects on human and animal health. Therefore, it is necessary to develop sensitive and reliable screening methods. In this study, a highly sensitive monoclonal antibody (MAb) against FB1, clone 2D7, was produced, and the 50% inhibition concentration (IC50) of the MAb was 2.2 ng/mL in buffer. The MAb showed high cross-reactivity with fumonisin B2 (FB2), and negligible cross-reactivity with other mycotoxins. A sensitive indirect competitive enzyme-linked immunosorbent assay (icELISA) based on this MAb was developed for the determination of FB1 in maize. In spiked samples (100, 200 and 500 μg/kg), the average recoveries ranged from 78 ± 11 to 107 ± 4%, and the coefficient of variation ranged from 3 to 15%. The limit of detection of the icELISA was 5.4 μg/kg. This method was compared to liquid chromatography tandem mass spectrometry (LC–MS/MS) using naturally contaminated samples, and the correlation coefficient was above 0.82. These results show the reliability of the icELISA method for the determination of FB1 in maize. [Copyright &y& Elsevier]