Established a new double antibodies sandwich enzyme-linked immunosorbent assay for detecting Bacillus thuringiensis (Bt) Cry1Ab toxin based single-chain variable fragments from a na茂ve mouse phage displayed library

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• 作者：Xiao Zhang ; Chongxin Xu ; Cunzheng Zhang ; Yuan Liu ; Yajing Xie ; Xianjin Liu ; ^{jaasliu@jaas.ac.cn" class="auth_mail}
• 关键词：Cry1Ab toxin ; ScFv ; Naï ; ve mouse phage displayed library ; Double antibodies sandwich immunoassay
• 刊名：Toxicon
• 出版年：April, 2014
• 年：2014
• 卷：81
• 期：Complete
• 页码：13-22
• 全文大小：871 K

文摘

ScFvs are composed of the variable regions of the heavy and light chains via a short linker that maintain the specific antigen binding abilities of antibodies. In this study, we constructed a na茂ve mouse phage displayed library to generate scFvs against Cry1Ab toxin. After affinity panning, positive phage-scFvs were isolated, sequenced and characterized by ELISA. The best binding ability scFv-G9 was expressed and purified. SDS-PAGE indicated that the relative molecular mass of scFv was estimated at 28聽kDa. The purified scFv-G9 was used to develop a new DAS-ELISA for detecting Cry1Ab toxin, within minimum detection limit of 0.008聽渭g聽mL^鈭?, a working range 0.018-6.23聽渭g聽mL^鈭?, and the linear curve displayed an acceptable correlation coefficient of 0.98. The cross-reactivity showed that scFv-G9 had strongly binding ability to Cry1Ac toxin, but not to Cry1B, Cry1C and Cry1F toxin. The average recoveries of Cry1Ab toxin from spiked leaf and rice samples were in the range 92.1-94.8%, and 91.6-98.6%, respectively, with a coefficient of variation (C.V) less than 5.0%. These results showed promising applications of scfv-G9 for detecting Cry1Ab toxin with new DAS-ELISA.