A phage-displayed chicken single-chain antibody fused to alkaline phosphatase detects Fusarium pathogens and their presence in cereal grains
A phage-displayed chicken single-chain antibody fused to alkaline phosphatase detects Fusarium pathogens and their presence in cereal grains
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与碱性磷酸酶融合的噬菌体展示鸡单链抗体可检测镰刀菌病原体及其在谷物中的存在
DOI：
10.1016/j.aca.2012.12.022
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发表时间：
2013-02-18
影响因子：
6.2
通讯作者：
Liao, Yu-Cai
中科院分区：
文献类型：
--
作者：
Hu, Zu-Quan;Li, He-Ping;Liao, Yu-Cai
镰刀菌属及其有毒真菌毒素分布于世界各地，在农业和食品安全方面特别重要。检测病原体的简单分析方法对于预测疾病和控制真菌毒素至关重要。本文介绍了一种利用单链抗体（scFv）和碱性磷酸酶（AP）融合的方法，用于方便和灵敏地检测镰刀菌病原体。一种特异于F.利用噬菌体展示技术从免疫鸡噬菌粒库中筛选出轮枝菌属的噬菌体。该抗体可与真菌孢子和菌丝体表面结合。轮枝状构建了scFv-AP融合蛋白，并在细菌中进行了可溶性表达。抗体性质和酶活性均被保留，并且通过添加接头增强融合物的抗原结合能力。表面等离子体共振测量证实，与融合物的亲本scFv抗体相比，融合物显示出4倍更高的亲和力。免疫印迹分析表明，融合蛋白与F.轮枝孢霉属（verticillioides），酶联免疫吸附试验表明，该真菌的检测限低于10（-2）μ g mL（-1），上级优于scFv抗体。融合蛋白能够检测真菌浓度低至10（-3）毫克克（-1）的玉米籽粒在自然和人工污染的样品。因此，该融合基因可用于田间、粮食或食品中镰刀菌污染的快速、简便诊断。(C)2012爱思唯尔有限公司版权所有。
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(-2) mu g mL(-1), superior to the scFv antibody. The fusion protein was able to detect fungal concentrations as low as 10(-3) mg g(-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. (C) 2012 Elsevier B.V. All rights reserved.