Surface Plasmon Resonance‐Based Immunoassay for the Detection of Aflatoxin B₁Using Single‐Chain Antibody Fragments

Aflatoxin B1 (AFB1) is a highly toxic secondary metabolite of the fungal species Aspergillus flavus and Aspergillus parasiticus produced under certain environmental conditions. The gene encoding an AFB1‐specific single‐chain fragment variable (scFv) was isolated from a pre‐immunized phage display library and used to express a monomeric and dimeric scFv, specific for AFB1, in Escherichia coli. The monomeric and dimeric scFv were then applied to the development of surface plasmon resonance‐based inhibition immunoassays for the detection of AFB1. Regeneration of the sensor surface, which consisted of a CM5 chip immobilized with an AFB1 derivative, was investigated and enabled at least 75 binding regeneration cycles. The inhibition assays developed had ranges of detection between 390 and 12,000 pg mL−1 (ppb) for the monomeric scFv and between 190 and 24,000 pg mL−1 (ppb) for the dimeric scFv, with coefficients of variation for the inter‐day variability studies ranging from 1.9–4.18% and 3–11.53%, respectively.