title: Deoxynivalenol-mimic nanobody isolated from a naïve phage display nanobody library and its application in immunoassay.
abstract: In this study, using mycotoxin deoxynivalenol (DON) as a model hapten, we developed a nanobody-based environmental friendly immunoassay for sensitive detection of DON. Two nanobodies (N-28 and N-31) which bind to anti-DON monoclonal antibody (MAb) were isolated from a naive phage display library. These nanobodies are clonable, thermally stable and mycotoxin-free products and can be served as coating antigen mimetics in heterologous immunoassay. The half inhibition concentration (IC50) of the immunoassay developed with N-28 and N-31 was 8.77 ± 0.41 ng mL(-1) and 19.97 ± 0.84 ng mL(-1), respectively, which were 18- and 8-fold more sensitive than the conventional coating antigen (DON-BSA) based immunoassay. In order to better understand the molecular mechanism of antigen mimicry by nanobody, the 3D structure of "nanobody (N-28) - anti-DON MAb" complex was presented and verified by molecular modeling and alanine-scanning mutagenesis. The results showed that hydrogen bond and hydrophobic interaction formed between Thr 102 - Ser 106 of N-28 and CDR H3 residues of anti-DON antibody may contribute to their binding. This novel concept of enhancing sensitivity of immunoassay for DON based on nanobody may provide potential applications in a general method for immunoassay of various food chemical contaminants.
authors: State Key Laboratory of Food Science and Technology, Sino-German Joint Research Institute, Nanchang University, No. 235 Nanjing East Road, Nanchang 330047, China.
authors: Yu-Lou
authors: YL
authors: Qiu
authors: State Key Laboratory of Food Science and Technology, Sino-German Joint Research Institute, Nanchang University, No. 235 Nanjing East Road, Nanchang 330047, China. Electronic address: heqinghua@ncu.edu.cn.
authors: Qing-Hua
authors: QH
authors: He
authors: State Key Laboratory of Food Science and Technology, Sino-German Joint Research Institute, Nanchang University, No. 235 Nanjing East Road, Nanchang 330047, China. Electronic address: xuyang@ncu.edu.cn.
authors: Yang
authors: Y
authors: Xu
authors: Molecular Food Microbiology Laboratory, Department of Food Science, Purdue University, West Lafayette, IN, USA.
authors: Arun K
authors: AK
authors: Bhunia
authors: State Key Laboratory of Food Science and Technology, Sino-German Joint Research Institute, Nanchang University, No. 235 Nanjing East Road, Nanchang 330047, China.
authors: Zhui
authors: Z
authors: Tu
authors: State Key Laboratory of Food Science and Technology, Sino-German Joint Research Institute, Nanchang University, No. 235 Nanjing East Road, Nanchang 330047, China.
authors: Bo
authors: B
authors: Chen
authors: State Key Laboratory of Food Science and Technology, Sino-German Joint Research Institute, Nanchang University, No. 235 Nanjing East Road, Nanchang 330047, China.
authors: Yuan-Yuan
authors: YY
authors: Liu
mesh_terms: D000595:Amino Acid Sequence; D000911:Antibodies, Monoclonal / Q000737:chemistry / Q000276:immunology; D002523:Edible Grain / Q000382:microbiology*; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods; D006241:Haptens / Q000032:analysis / Q000276:immunology; D007118:Immunoassay / Q000379:methods*; D062105:Molecular Docking Simulation; D018716:Molecular Mimicry; D008969:Molecular Sequence Data; D009183:Mycotoxins / Q000032:analysis* / Q000276:immunology; D019151:Peptide Library; D061905:Single-Domain Antibodies / Q000737:chemistry* / Q000276:immunology; D014255:Trichothecenes / Q000032:analysis* / Q000276:immunology
chemical_list: D000911:Antibodies, Monoclonal; D006241:Haptens; D009183:Mycotoxins; D019151:Peptide Library; D061905:Single-Domain Antibodies; D014255:Trichothecenes; C007262:deoxynivalenol
keywords: Deoxynivalenol; Immunoassay; Mimetics; Nanobody