A phage-displayed chicken single-chain antibody fused to alkaline phosphatase detects Fusarium pathogens and their presence in cereal grains

A phage-displayed chicken scFv antibody, FvSG7, binds on the surface antigen of conidiospores and the mycelia of F. verticillioides. Its fusion with alkaline phosphatase (AP) through a 218 linker displayed a 4-fold higher affinity compared with the parent scFv antibody and efficiently detected toxig...

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Bibliographic Details
Published in:Analytica chimica acta 2013-02, Vol.764, p.84-92
Main Authors: Hu, Zu-Quan, Li, He-Ping, Zhang, Jing-Bo, Huang, Tao, Liu, Jin-Long, Xue, Sheng, Wu, Ai-Bo, Liao, Yu-Cai
Format: Article
Language:English
Subjects:
Alkaline phosphatase
Alkaline Phosphatase - genetics
Alkaline Phosphatase - metabolism
Amino Acid Sequence
Animals
Antibodies
Antibody Affinity
Bacteria
Chickens
Edible Grain - microbiology
Enzyme-Linked Immunosorbent Assay
Food Contamination - analysis
Fungi
Fusarium
Fusarium - metabolism
Fusarium pathogens
Fusion protein
Grains
Immunoblotting
Kinetics
Mathematical analysis
Molecular Sequence Data
Mycotoxins
Mycotoxins - analysis
Pathogens
Peptide Library
Phage display
Recombinant Fusion Proteins - biosynthesis
Recombinant Fusion Proteins - genetics
Recombinant Fusion Proteins - immunology
Single-Chain Antibodies - genetics
Single-Chain Antibodies - immunology
Single-Chain Antibodies - metabolism
Single-chain variable fragment
Zea mays
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Summary:A phage-displayed chicken scFv antibody, FvSG7, binds on the surface antigen of conidiospores and the mycelia of F. verticillioides. Its fusion with alkaline phosphatase (AP) through a 218 linker displayed a 4-fold higher affinity compared with the parent scFv antibody and efficiently detected toxigenic Fusarium pathogens in cereal grains. [Display omitted] ► Generation of a highly reactive scFv antibody against F. verticillioides. ► Localization of the antibody binding to the surface target of F. verticillioides. ► Expression of the antibody–alkaline phosphatase (AP) fusion linked by a 218 linker. ► The antibody–AP fusion has a higher affinity than the parental antibody. ► The antibody–AP fusion detects toxigenic Fusarium pathogens in cereal grains. Fusarium and its poisonous mycotoxins are distributed worldwide and are of particular interest in agriculture and food safety. A simple analytical method to detect pathogens is essential for forecasting diseases and controlling mycotoxins. This article describes a proposed method for convenient and sensitive detection of Fusarium pathogens that uses the fusion of single-chain variable fragment (scFv) and alkaline phosphatase (AP). A highly reactive scFv antibody specific to soluble cell wall-bound proteins (SCWPs) of F. verticillioides was selected from an immunized chicken phagemid library by phage display. The antibody was verified to bind on the surface of ungerminated conidiospores and mycelia of F. verticillioides. The scFv–AP fusion was constructed, and soluble expression in bacteria was confirmed. Both the antibody properties and enzymatic activity were retained, and the antigen-binding capacity of the fusion was enhanced by the addition of a linker. Surface plasmon resonance measurements confirmed that the fusion displayed 4-fold higher affinity compared with the fusion's parental scFv antibody. Immunoblot analyses showed that the fusion had good binding capacity to the components from SCWPs of F. verticillioides, and enzyme-linked immunosorbent assays revealed that the detection limit of the fungus was below 10−2μgmL−1, superior to the scFv antibody. The fusion protein was able to detect fungal concentrations as low as 10−3mgg−1 of maize grains in both naturally and artificially contaminated samples. Thus, the fusion can be applied in rapid and simple diagnosis of Fusarium contamination in field and stored grain or in food.
ISSN:0003-2670
1873-4324
DOI:10.1016/j.aca.2012.12.022