Aflatoxins (AFs) including AFB
1, AFB
2, AFG
1 and AFG
2 are widely found in agriculture products, and AFB
1 is considered one of the most toxic and harmful mycotoxins. Herein, a highly sensitive (at the pg mL
−1 level)
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Aflatoxins (AFs) including AFB
1, AFB
2, AFG
1 and AFG
2 are widely found in agriculture products, and AFB
1 is considered one of the most toxic and harmful mycotoxins. Herein, a highly sensitive (at the pg mL
−1 level) and group-specific enzyme-linked immunosorbent assay (ELISA) for the detection of AFB
1 in agricultural and aquiculture products was developed. The AFB
1 derivative containing a carboxylic group was synthesized and covalently linked to bovine serum albumin (BSA). The AFB
1-BSA conjugate was used as an immunogen to immunize mice. A high-quality monoclonal antibody (mAb) against AFB
1 was produced by hybridoma technology, and the mAb-based ELISA for AFB
1 was established. IC
50 and limit of detection (LOD) of the ELISA for AFB
1 were 90 pg mL
−1 and 18 pg mL
−1, respectively. The cross-reactivities (CRs) of the assay with AFB
2, AFG
1, and AFG
2 were 23.6%, 42.5%, and 1.9%, respectively, revealing some degree of group specificity. Corn flour, wheat flour, and crab roe samples spiked with different contents of AFB
1 were subjected to ELISA procedures. The recoveries and relative standard deviation (RSD) of the ELISA for AFB
1 in spiked samples were 78.3–116.6% and 1.49–13.21% (
n = 3), respectively. Wheat flour samples spiked with the mixed AF (AFB
1, AFB
2, AFG
1, AFG
2) standard solution were measured by ELISA and LC-MS/MS simultaneously. It was demonstrated that the proposed ELISA can be used as a screening method for evaluation of AFs (AFB
1, AFB
2, AFG
1, AFG
2) in wheat flour samples.
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