Development of a specific anti-idiotypic nanobody for monitoring aflatoxin M1 in milk and dairy products

Anti-idiotypic antibodies (AIds) display stable protein conformation, offering an alternative to conventional antigens in immunoassays. In this work, an anti-idiotypic variable domain of heavy-chain antibody (VHH) or nanobody named VHH C4 binding to monoclonal antibody (mAb) 2C9 against aflatoxin M 1 (AFM 1 ) was screened out from an alpaca immunized phage-displayed library. VHH C4 shows outstanding features in specificity and sensitivity, the IC 50 is 0.25 ng/mL and exhibit almost no cross-reactivity with other aflatoxin analogs. As our knowledge, it is the first report for developing an anti-idiotypic nanobody for AFM 1 . An environment-friendly competitive ELISA was developed using VHH C4 as a coating antigen. The results revealed a linear range from 0.10 ng/mL to 0.60 ng/mL, with a limit of detection (LOD) of 0.05 ng/mL. The recovery of spiked samples (pure milk, yogurt, and milk powder) ranged from 84.1% to 119.1%. The ELISA results of AFM 1 content in naturally contaminated samples were in good agreement with those determined by high performance liquid chromatography (HPLC). The results demonstrated that the nanobody VHH C4 is a promising alternative antigen in developing immunological methods for the detection of AFM 1 in dairy products.