A Rapid Aflatoxin B1 ELISA:  Development and Validation with Reduced Matrix Effects for Peanuts, Corn, Pistachio, and Soybeans

Among the competitive ELISAs for aflatoxins that have been described, few have been adequately validated for reduced matrix effects. Using an aflatoxin B1 (AFB1)-specific polyclonal antibody (produced from AFB1−oxime conjugated to bovine serum albumin (BSA)) and AFB1− and AFB2−enzyme conjugates, four direct competitive ELISAs based on 96-microwell plates (two standard assays and two rapid assays) were developed, paying special attention to producing a robust assay relatively free of interferences for a range of agricultural products. The antibody was AFB1-specific, detecting only AFB1 in a mixture of four aflatoxins (AFB1, AFB2, AFG1, and AFG2), but showed significant cross-reaction with AFG1 (57−61%) when an individual compound was tested. Standard assays (long assays) exhibited higher sensitivities than rapid assays (short assays) with IC50 values of 12 ± 1.5 and 9 ± 1.5 μg/kg in sample (with 1 in 5 dilution of sample extract) for AFB1 and AFB2−enzyme conjugates, respectively. These assays have narrower detection ranges (7.1−55.5 μg/kg in sample) and required dilution of sample extracts to overcome solvent and matrix interferences, making these assays less ideal as analytical methods. Rapid assays exhibited IC50 values of 21.6 ± 2.7 and 12 μg/kg in sample for AFB1− and AFB2−enzyme conjugates, respectively. These assays have ideally broader detection ranges (4.2−99.9 μg/kg in sample) and showed no methanol effects up to 80% with significantly reduced matrix interferences as a result of the shorter incubation times and increasing the amounts of enzyme conjugate used. Therefore, the rapid assays were formatted to perform without a need for extract dilution. The rapid assays can be completed within 15 min, potentially suitable for receival bays where quick decision-making to segregate low and high contamination is critical. Further validation using the rapid assay with AFB1−enzyme conjugate indicated relatively good recoveries of AFB1 spiked in corn, peanuts, pistachio, and soybeans, which were free from significant matrix effects. It can be concluded that this rapid assay would be suitable for monitoring aflatoxin AFB1 at current legal maximum residue limits of 10 μg/kg in food such as corn, peanuts, pistachio, and soybeans. Keywords: Aflatoxin B1; ELISA; food; antibodies; carcinogen; monitoring; food safety