title: Modification of a deoxynivalenol-antigen-mimicking nanobody to improve immunoassay sensitivity by site-saturation mutagenesis.
abstract: A nanobody (N-28) which can act as a deoxynivalenol (DON) antigen has been generated, and its residues Thr102-Ser106 were identified to bind with anti-DON monoclonal antibody by alanine-scanning mutagenesis. Site-saturation mutagenesis was used to analyze the plasticity of five residues and to improve the sensitivity of the N-28-based immunoassay. After mutagenesis, three mutants were selected by phage immunoassay and were sequenced. The half-maximal inhibitory concentrations of the immunoassay based on mutants N-28-T102Y, N-28-V103L, and N-28-Y105F were 24.49 ± 1.0, 51.83 ± 2.5, and 35.65 ± 1.6 ng/mL, respectively, showing the assay was, respectively, 3.2, 1.5, and 2.2 times more sensitive than the wild-type-based assay. The best mutant, N-28-T102Y, was used to develop a competitive phage ELISA to detect DON in cereals with high specificity and accuracy. In addition, the structural properties of N-28-T102Y and N-28 were investigated, revealing that the affinity of N-28-T102Y decreased because of increased steric hindrance with the large side chain. The lower-binding-affinity antigen mimetic may contribute to the improvement of the sensitivity of competitive immunoassays. These results demonstrate that nanobodies would be a favorable tool for engineering. Moreover, our results have laid a solid foundation for site-saturation mutagenesis of antigen-mimicking nanobodies to improve immunoassay sensitivity for small molecules.
authors.affiliation: State Key Laboratory of Food Science and Technology, Sino-German Joint Research Institute, Nanchang University, No. 235 Nanjing East Road, Nanchang, 330047, China.
authors.forename: Yu-Lou
authors.initials: YL
authors.lastname: Qiu
authors.affiliation: State Key Laboratory of Food Science and Technology, Sino-German Joint Research Institute, Nanchang University, No. 235 Nanjing East Road, Nanchang, 330047, China. heqinghua@ncu.edu.cn.
authors.forename: Qing-Hua
authors.initials: QH
authors.lastname: He
authors.affiliation: State Key Laboratory of Food Science and Technology, Sino-German Joint Research Institute, Nanchang University, No. 235 Nanjing East Road, Nanchang, 330047, China. xuyang@ncu.edu.cn.
authors.forename: Yang
authors.initials: Y
authors.lastname: Xu
authors.affiliation: State Key Laboratory of Food Science and Technology, Sino-German Joint Research Institute, Nanchang University, No. 235 Nanjing East Road, Nanchang, 330047, China.
authors.forename: Wei
authors.initials: W
authors.lastname: Wang
authors.affiliation: State Key Laboratory of Food Science and Technology, Sino-German Joint Research Institute, Nanchang University, No. 235 Nanjing East Road, Nanchang, 330047, China.
authors.forename: Yuan-Yuan
authors.initials: YY
authors.lastname: Liu
mesh_terms: D000941:Antigens / Q000737:chemistry* / Q000235:genetics / Q000276:immunology; D007118:Immunoassay / Q000295:instrumentation* / Q000379:methods; D018716:Molecular Mimicry; D016296:Mutagenesis; D057127:Single-Chain Antibodies / Q000737:chemistry* / Q000235:genetics* / Q000276:immunology; D014255:Trichothecenes / Q000737:chemistry* / Q000235:genetics / Q000276:immunology
chemical_list: D000941:Antigens; D057127:Single-Chain Antibodies; D014255:Trichothecenes; C007262:deoxynivalenol
keywords: Deoxynivalenol; Immunoassay; Mimetics; Nanobody; Site-saturation mutagenesis