Nanobody-Based Enzyme Immunoassay for Aflatoxin in Agro-Products with High Tolerance to Cosolvent Methanol

A phage-displayed library of variable domain of heavy chain of the heavy chain antibody (VHH) or nanobody (Nb) was constructed after immunizing an alpaca with aflatoxin B1 (AFB1) conjugated with bovine serum albumin (AFB1–BSA). Two AFB1-specific nanobodies were selected. The obtained nanobodies were compared to an aflatoxin-specific monoclonal antibody B5 with respect to stability under organic solvents and high temperature. The two nanobodies could bind antigen specifically after exposure to temperatures as high as 95 °C. Besides, the nanobodies showed better or similar tolerance to organic solvents. A competitive ELISA with nanobody Nb26 was developed for the analysis of AFB1, exhibiting an IC50 value of 0.754 ng/mL (2.4 μM), linear range from 0.117 to 5.676 ng/mL. Due to the high tolerance to methanol, sample extracts were analyzed by nanobody-based ELISA without dilution. The recovery from spiked peanut, rice, corn and feedstuff ranged from 80 to 115%. In conclusion, the isolated nanobodies are excellent candidates for immunoassay application in aflatoxin determination.