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Aspergillus  and its poisonous mycotoxins are distributed worldwide throughout the environment and are of particular interest in agriculture and food safety. In order to develop a specific method for rapid detection of  Aspergillus flavus  to forecast diseases and control aflatoxins, a nanobody, PO8-VHH, highly reactive to  A. flavus  was isolated from an immunized alpaca nanobody library by phage display. The nanobody was verified to bind to the components of extracellular and intracellular antigen from both  A. flavus  and  A. parasiticus . To construct a sandwich format immunoassay, polyclonal antibodies against  Aspergillus  were raised with rabbits. Finally, a highly selective nanobody-polyclonal antibody sandwich enzyme-linked immunosorbent assay was optimized and developed. The results revealed that the detection limits of the two fungi were as low as 1 μg mL −1 , and that it is able to detect fungal concentrations below to 2 μg mg −1  of peanut and maize grains in both artificially and naturally contaminated samples. Therefore, we here provided a rapid and simple method for monitoring  Aspergillus  spp. contamination in agricultural products.

Determination of Aspergillus pathogens in agricultural products by a specific nanobody-polyclonal antibody sandwich ELISA