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

免疫分析 异源的 效价 免疫原 化学 牛血清白蛋白 生物 分子生物学 抗体 色谱法 生物化学 单克隆抗体 基因 免疫学
作者
Weixuan Wang,Gan Gu,Ruya Yin,Jiajin Fu,Mingpeng Jing,Zhen Shen,Daowan Lai,Baomin Wang,Ligang Zhou
出处
期刊:Toxins [MDPI AG]
卷期号:14 (10): 659-659 被引量:9
标识
DOI:10.3390/toxins14100659
摘要

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.
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