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.

Aflatoxins are a group of extremely toxic small molecules that have been involved in human hepatic and extrahepatic carcinogenesis as causative agents. Herein, we developed a real-time immuno polymerase chain reaction (IPCR) assay for the accurately quantitative detection of aflatoxins in agri-products base on a M13 phage containing aflatoxin anti-idiotypic nanobody and its encoding DNA which was used to design the specific primers. The limit of detection (LOD) of the assay is 0.02 ng/mL, which exhibits a 4-fold improvement over traditional phage ELISA. The developed method was successfully validated with the samples of corn, rice, peanut, and feedstuff, which are major aflatoxin-contaminated agri-products. And the recoveries were from 77.05 to 122.16%. For further validation, the developed assay was also compared with a reference HPLC method for the analysis of aflatoxins in corn and peanuts, and concordant results (R(2) = 0.991) were obtained. In this context, this study provides a novel opportunity to analyze aflatoxins in agri-products.

A highly-sensitive TRFICA was developed to detect AFM1 in raw milk samples within 6 minutes without any sample pretreatments.