A Highly Sensitive and Group-Specific Enzyme-Linked Immunosorbent Assay (ELISA) for the Detection of AFB1 in Agriculture and Aquiculture Products

Abstract

Aflatoxins (AFs) including AFB₁, AFB₂, AFG₁ and AFG₂ are widely found in agriculture products, and AFB₁ 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 AFB₁ in agricultural and aquiculture products was developed. The AFB₁ derivative containing a carboxylic group was synthesized and covalently linked to bovine serum albumin (BSA). The AFB₁-BSA conjugate was used as an immunogen to immunize mice. A high-quality monoclonal antibody (mAb) against AFB₁ was produced by hybridoma technology, and the mAb-based ELISA for AFB₁ was established. IC₅₀ and limit of detection (LOD) of the ELISA for AFB₁ were 90 pg mL^-1 and 18 pg mL^-1, respectively. The cross-reactivities (CRs) of the assay with AFB₂, AFG₁, and AFG₂ 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 AFB₁ were subjected to ELISA procedures. The recoveries and relative standard deviation (RSD) of the ELISA for AFB₁ in spiked samples were 78.3-116.6% and 1.49-13.21% (n = 3), respectively. Wheat flour samples spiked with the mixed AF (AFB₁, AFB₂, AFG₁, AFG₂) 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 (AFB₁, AFB₂, AFG₁, AFG₂) in wheat flour samples.

Keywords: AFB1; aflatoxins; agricultural and aquiculture products; enzyme-linked immunosorbent assay (ELISA); monoclonal antibody (mAb); total evaluation.

Figure 1

MALDI-TOF-MS spectra of BSA and AFB₁-BSA. The m/z values of BSA and AFB₁-BSA are 65,269 and 67,604, respectively.

Figure 2

Standard curve of the ELISA for AFB₁ detection under optimal experimental conditions. The bars represent the standard derivations of the values of B/B₀ × 100% (n = 5). The IC₅₀ was 90 pg mL⁻¹, and the LOD was 18 pg mL⁻¹.