Isolation of single chain variable fragment (scFv) specific for Cry1C toxin from human single fold scFv libraries

详细信息    查看全文

• 作者：Yun Wang ; Xiao Zhang ; Cunzheng Zhang ; Yuan Liu ; Xianjin Liu
• 关键词：Cry1C ; scFv ; Phage displayed library ; Immunoassay
• 刊名：Toxicon
• 出版年：2012
• 出版时间：1 December, 2012
• 年：2012
• 卷：60
• 期：7
• 页码：1290-1297
• 全文大小：461 K

文摘

As bioinsecticides Bacillus thuringiensis Cry1C ¦Ä-endotoxins also have been used in genetically modified crops worldwide since last century. In this study, single chain variable fragments (scFvs), which could specifically recognize and detect Cry1C in food samples, were isolated from naive phage displayed human antibody libraries (Tomlinson I?+?J) by iterative affinity selection procedure instead of immunization process. With increasing selection pressure, after four rounds of panning, three individual scFvs were obtained and sequenced. The antibodies were characterized by enzyme-linked immunosorbent assay (ELISA). Thereafter, a conformed novel anti-Cry1C scFv, namely scFv-H6, was expressed in Escherichia coli (E. coli) HB2151 and purified by Ni metal ion affinity chromatography. An indirect competitive ELISA assay (ic-ELISA) of scFv-H6 was developed for the determination of Cry1C toxin in the range from 0.023?¦Ìg?mL^?1 to 4.35?¦Ìg?mL^?1, and 50 % inhibition concentration (IC₅₀) was 0.39?¦Ìg?mL^?1. This approach showed ignorable cross-reactivity with toxin Cry1Ac and Cry1B (3.51 % and 7.28 % , respectively). This ic-ELISA approach was exploited for the determination of Cry1C in spiked ground rice samples with a mean recovery rate of 92.5 % and coefficient of variation (C.V.) less than 5.0 % . This study proves that phage display libraries provide a valuable system for the low-cost, rapid and continuous generation of specific antibody fragments directed against toxin targets and develop a simple detection method. Our results show that anti-Cry1C scFv could be a valuable tool for detection of Cry1C in food and agricultural samples.