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

摘要

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 (AFB_1) conjugated with bovine serum albumin (AFB_1-BSA). Two AFB_1-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℃. Besides, the nanobodies showed better or similar tolerance to organic solvents. A competitive ELISA with nanobody Nb26 was developed for the analysis of AFB_1, exhibiting an IC_(50) 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.