Rapid isolation of single-chain antibodies from a human synthetic phage display library for detection of Bacillus thuringiensis (Bt) Cry1B toxin

文献类型： 外文期刊

第一作者： Zhang, Xiao

作者： Zhang, Xiao;Liu, Yuan;Zhang, Cunzheng;Wang, Yun;Liu, Xianjin;Zhang, Xiao;Wang, Yun;Xu, Chongxin

作者机构：

关键词： Cry1B toxin;Scfv;Phage displayed library;Immunoassay

期刊名称：ECOTOXICOLOGY AND ENVIRONMENTAL SAFETY （ 影响因子：6.291； 五年影响因子：6.393 ）

ISSN： 0147-6513

年卷期： 2012 年 81 卷

页码：

收录情况： SCI

摘要： Single chain variable fragment antibody (scFv) is capable of binding its target antigens and is one of the most popular recombinant antibodies format for many applications. In this study, a large human synthetic phage displayed library (Tomlinson J) was employed to generate scFvs against Cry1B toxin by affinity panning. After four rounds of panning, six monoclonal phage particles capable of binding with the Cry1B were isolated, sequenced and characterized by Enzyme-Linked Immunosorbent Assay (ELISA). Two of the identified novel anti-Cry1B scFvs, namely H9 and B12, were expressed in Escherichia coli HB2151 and purified by Ni metal ion affinity chromatography. Sodium dodecyl sulfate polyacrylamine gel electrophoresis (SDS-PAGE) indicated that the relative molecular mass of scFv was estimated at 30 kDa. The purified scFv-H9 was used to develop an indirect competitive enzyme-linked immunosorbent assay (ic-ELISA) for Cry1B toxin. The linear range of detection for standards in this ic-ELISA was approximately 0.19-1.1 mu g mL(-1) and 50% inhibition of control (IC50) was 0.84 mu g mL(-1) for Cry1B. The affinity of scfv-H9 was (1.95 +/- 0.12) x 10(7) M-1 and showed cross-reactivity with Cry1Ab toxin and Cry1Ac toxin (8.53% and 7.58%, respectively), higher cross-reactivity (12.8%) with Cry1C toxin. The average recoveries of Cry1B toxin from spiked leaf and rice samples were in the range 89.5-96.4%, and 88.5-95.6%, respectively, with a coefficient of variation (C.V) less than 6.0%. These results showed promising applications of scfv-H9 for detecting Cry1B toxin in agricultural and environmental samples. (C) 2012 Elsevier Inc. All rights reserved.

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