A Nanobody-Based Immunoassay for Detection of Ustilaginoidins in Rice Samples

Ustilaginoidins are a class of bis-naphtho-γ-pyrone mycotoxins produced by the pathogen Villosiclava virens of rice false smut, which has recently become one of the most devastating diseases in rice-growing regions worldwide. In this research, the nanobody phage display library was established after an alpaca was immunized with the hemiustilaginoidin F-hapten coupled with bovine serum albumin (BSA). Heterologous antigen selection and combing trypsin with competition alternant elution methods were performed for nanobody screening. Two nanobodies, namely, Nb-B15 and Nb-C21, were selected for the establishment of indirect competitive enzyme-linked immunosorbent assays (ic-ELISAs). For Nb-B15 and Nb-C21, their IC₅₀ values were 11.86 μg/mL and 11.22 μg/mL, and the detection ranges were at 3.41-19.98 μg/mL and 1.17-32.13 μg/mL, respectively. Two nanobodies had a broad spectrum to quantify the contents of total ustilaginoidins in rice samples according to cross-reactivity. The recognition mechanisms of Nb-B15 and Nb-C21 against ustilaginoidin A were elucidated by molecular modeling and docking. The key amino acid sites for the binding of Nb-B15 or Nb-C21 to ustilaginoidin A were mainly located in the FR1 and CDR1 regions. As Nb-B15 was superior to Nb-C21 in the aspects of protein expression, ELISA titer, and tolerance to organic solvents, it was selected for application in the detection of actual contaminated rice samples. The total ustilaginoidin contents of rice samples were analyzed by Nb-B15-based ic-ELISA and HPLC-DAD, between which the results were found to be consistent. The developed immunoassay based on the nanobody from the alpaca can be employed as a rapid and effective method for detection of total utilaginoidins in contaminated rice samples.