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p Uniform Orientation of Biotinylated Nanobody as an Affinity Binder for Detection of Bacillus thuringiensis (Bt) Cry1Ac Toxin

文献类型： 外文期刊

作者： Li, Min 1 ; Zhu, Min 1 ; Zhang, Cunzheng 2 ; Liu, Xianjin 2 ; Wan, Yakun 1 ;

作者机构： 1.Southeast Univ, Inst Life Sci, Minist Educ, Key Lab Dev Genes & Human Dis, Nanjing 210096, Jiangsu, Peoples R China

2.Jiangsu Acad Agr Sci, Inst Food Safety, Nanjing 210014, Peoples R China

3.Jiangsu Nanobody Engn & Res Ctr, Nantong 226010, Peoples R China

关键词： nanobody;Cry1Ac toxin;streptavidin;DAS-ELISA

期刊名称：TOXINS （ 影响因子：4.546； 五年影响因子：4.8 ）

ISSN： 2072-6651

年卷期： 2014 年 6 卷 12 期

页码：

收录情况： SCI

摘要： Nanobodies are the smallest natural fragments with useful properties such as high affinity, distinct paratope and high stability, which make them an ideal tool for detecting target antigens. In this study, we generated and characterized nanobodies against the Cry1Ac toxin and applied them in a biotin-streptavidin based double antibodies (nanobodies) sandwich-ELISA (DAS-ELISA) assay. After immunizing a camel with soluble Cry1Ac toxin, a phage displayed library was constructed to generate Nbs against the Cry1Ac toxin. Through successive rounds of affinity bio-panning, four nanobodies with greatest diversity in CDR3 sequences were obtained. After affinity determination and conjugating to HRP, two nanobodies with high affinity which can recognize different epitopes of the same antigen (Cry1Ac) were selected as capture antibody (Nb61) and detection antibody (Nb44). The capture antibody (Nb61) was biotinylated in vivo for directional immobilization on wells coated with streptavidin matrix. Both results of specificity analysis and thermal stability determination add support for reliability of the following DAS-ELISA with a minimum detection limit of 0.005 mu g.mL(-1) and a working range 0.010-1.0 mu g.mL(-1). The linear curve displayed an acceptable correlation coefficient of 0.9976. These results indicated promising applications of nanobodies for detection of Cry1Ac toxin with biotin-streptavidin based DAS-ELISA system.

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[1]Bactrian camel nanobody-based immunoassay for specific and sensitive detection of Cry1Fa toxin. Wang, Pingyan,Li, Guanghui,Yan, Junrong,Wan, Yakun,Wan, Yakun,Hu, Yonghong,Zhang, Cunzheng,Liu, Xianjin.

[2]Establishment of a sandwich enzyme-linked immunosorbent assay for specific detection of Bacillus thuringiensis (Bt) Cry1Ab toxin utilizing a monoclonal antibody produced with a novel hapten designed with molecular model. Dong, Sa,Zhang, Xiao,Liu, Yuan,Zhang, Cunzheng,Xie, Yajing,Zhong, Jianfeng,Xu, Chongxin,Liu, Xianjin,Dong, Sa,Liu, Xianjin.

[3]Simultaneous production of monoclonal antibodies against Bacillus thuringiensis (Bt) Cry1 toxins using a mixture immunization. Dong, Sa,Zhang, Cunzheng,Liu, Yuan,Zhang, Xiao,Xie, Yajing,Zhong, Jianfeng,Xu, Chongxin,Liu, Xianjin,Dong, Sa,Liu, Xianjin. 2017

[4]Establishment of a sensitive time-resolved fluoroimmunoassay for detection of Bacillus thuringiensis Cry1Ie toxin based nanobody from a phage display library. Xu, Chongxin,Zhang, Cunzheng,Zhang, Xiao,Zhong, Jianfeng,Liu, Yuan,Hu, Xiaodan,Lin, Manman,Liu, Xianjin,Liu, Xiaoqin. 2017

[5]单链抗体对Cry1A类毒素的检测方法建立及识别差异初步研究. 曲婷婷,张霄,董飒,刘贝贝,李盼,王耘,张存政,刘贤金. 2019

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