title: Anti-idiotypic VHH phage display-mediated immuno-PCR for ultrasensitive determination of mycotoxin zearalenone in cereals.
abstract: Immunoassay is frequently used to analyze mycotoxin contamination. However, the introduction of mycotoxins or their conjugates in conventional immunoassay threatens the safety of individuals and the environment. The variable domain of heavy-chain antibodies (VHHs) can be used as alternative compounds to produce anti-idiotypic antibodies, which work as non-toxic surrogate reagents in immunoassay. In this work, anti-zearalenone (ZEN) monoclonal antibody (mAb) was used as the target for biopanning anti-idiotypic VHH from a naïve alpaca VHH phage display library. After four panning cycles, one anti-idiotypic VHH phage clone (Z1) was isolated and the Z1 based phage ELISA for ZEN showed a half inhibitory concentration (IC50) of 0.25±0.02ng/mL, a linear range of 0.11-0.55ng/mL, and a limit of detection (LOD) of 0.08ng/mL. Furthermore, the phage particles of Z1 were also applied to immuno-polymerase chain reaction (PD-IPCR), which supplied both the detection antigens and deoxyribonucleic acid (DNA) templates. Compared with that of phage ELISA, the LOD of Z1 based PD-IPCR was 12-fold improved, with a detection limit of 6.5pg/mL and a linear range of 0.01-100ng/mL. The proposed method was then validated with liquid chromatography-tandem mass spectrometry (LC-MS/MS). Results showed the reliability of PD-IPCR for the determination of ZEN in cereal samples. The use of anti-idiotypic VHH phage as non-toxic surrogate and signal-amplification function of PCR make it a promising method for actual ZEN analysis in cereals.
authors.affiliation: College of Life Science, Nanchang University, No. 999 Xuefu Road, Nanchang 330031, People's Republic of China; State Key Laboratory of Food Science and Technology and, Nanchang University, No. 235 Nanjing East Road, Nanchang 330047, People's Republic of China; Sino-German Joint Research Institute, Nanchang University, No. 235 Nanjing East Road, Nanchang 330047, People's Republic of China.
authors.forename: Xianxian
authors.initials: X
authors.lastname: Wang
authors.affiliation: Sino-German Joint Research Institute, Nanchang University, No. 235 Nanjing East Road, Nanchang 330047, People's Republic of China. Electronic address: heqinghua@ncu.edu.cn.
authors.forename: Qinghua
authors.initials: Q
authors.lastname: He
authors.affiliation: State Key Laboratory of Food Science and Technology and, Nanchang University, No. 235 Nanjing East Road, Nanchang 330047, People's Republic of China; Sino-German Joint Research Institute, Nanchang University, No. 235 Nanjing East Road, Nanchang 330047, People's Republic of China.
authors.forename: Yang
authors.initials: Y
authors.lastname: Xu
authors.affiliation: State Key Laboratory of Food Science and Technology and, Nanchang University, No. 235 Nanjing East Road, Nanchang 330047, People's Republic of China; Sino-German Joint Research Institute, Nanchang University, No. 235 Nanjing East Road, Nanchang 330047, People's Republic of China.
authors.forename: Xing
authors.initials: X
authors.lastname: Liu
authors.affiliation: State Key Laboratory of Food Science and Technology and, Nanchang University, No. 235 Nanjing East Road, Nanchang 330047, People's Republic of China; Sino-German Joint Research Institute, Nanchang University, No. 235 Nanjing East Road, Nanchang 330047, People's Republic of China.
authors.forename: Mei
authors.initials: M
authors.lastname: Shu
authors.affiliation: State Key Laboratory of Food Science and Technology and, Nanchang University, No. 235 Nanjing East Road, Nanchang 330047, People's Republic of China.
authors.forename: Zhui
authors.initials: Z
authors.lastname: Tu
authors.affiliation: Sino-German Joint Research Institute, Nanchang University, No. 235 Nanjing East Road, Nanchang 330047, People's Republic of China.
authors.forename: Yanping
authors.initials: Y
authors.lastname: Li
authors.affiliation: State Key Laboratory of Food Science and Technology and, Nanchang University, No. 235 Nanjing East Road, Nanchang 330047, People's Republic of China; Sino-German Joint Research Institute, Nanchang University, No. 235 Nanjing East Road, Nanchang 330047, People's Republic of China.
authors.forename: Wei
authors.initials: W
authors.lastname: Wang
authors.affiliation: State Key Laboratory of Food Science and Technology and, Nanchang University, No. 235 Nanjing East Road, Nanchang 330047, People's Republic of China; Sino-German Joint Research Institute, Nanchang University, No. 235 Nanjing East Road, Nanchang 330047, People's Republic of China.
authors.forename: Dongmei
authors.initials: D
authors.lastname: Cao
mesh_terms: D000595:Amino Acid Sequence; D000888:Antibodies, Anti-Idiotypic / Q000737:chemistry / Q000235:genetics / Q000276:immunology*; D000911:Antibodies, Monoclonal / Q000737:chemistry / Q000235:genetics / Q000276:immunology; D002523:Edible Grain / Q000737:chemistry*; D004797:Enzyme-Linked Immunosorbent Assay; D005506:Food Contamination / Q000032:analysis*; D007143:Immunoglobulin Heavy Chains / Q000737:chemistry / Q000235:genetics / Q000276:immunology*; D057230:Limit of Detection; D008969:Molecular Sequence Data; D019151:Peptide Library; D016133:Polymerase Chain Reaction; D016415:Sequence Alignment; D012997:Solvents / Q000737:chemistry; D015025:Zearalenone / Q000032:analysis* / Q000276:immunology
chemical_list: D000888:Antibodies, Anti-Idiotypic; D000911:Antibodies, Monoclonal; D007143:Immunoglobulin Heavy Chains; D019151:Peptide Library; D012997:Solvents; D015025:Zearalenone
keywords: Anti-idiotypic antibody; Immuno-polymerase chain reaction; Zearalenone