Department of Entomology, College of Plant Protection, Nanjing Agricultural University, Key Laboratory of Monitoring and Management of Crop Diseases and Pest Insects, Ministry of Agriculture, Nanjing 210095, PR China.
Toxicon. 2012 Dec 1;60(7):1290-7. doi: 10.1016/j.toxicon.2012.08.014. Epub 2012 Sep 5.
As bioinsecticides Bacillus thuringiensis Cry1C δ-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 μg mL⁻¹ to 4.35 μg mL⁻¹, and 50% inhibition concentration (IC₅₀) was 0.39 μg 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.
作为生物杀虫剂，苏云金芽孢杆菌 Cry1C δ-内毒素自上世纪以来也已在全球范围内的转基因作物中使用。在这项研究中，通过迭代亲和选择程序（而不是免疫过程）从原始噬菌体展示人抗体文库（Tomlinson I + J）中分离出能够特异性识别和检测食品样品中的 Cry1C 的单链可变片段（scFv）。随着选择压力的增加，经过四轮淘选，获得了三个单独的 scFv 并进行了测序。使用酶联免疫吸附测定（ELISA）对抗体进行了表征。此后，在大肠杆菌（E. coli）HB2151 中表达并通过 Ni 金属离子亲和层析纯化了一种新的 Cry1C 特异性 scFv，即 scFv-H6。开发了用于测定 Cry1C 毒素的间接竞争 ELISA（ic-ELISA），其范围为 0.023 μg mL⁻¹ 至 4.35 μg mL⁻¹，50%抑制浓度（IC₅₀）为 0.39 μg mL⁻¹。该方法与毒素 Cry1Ac 和 Cry1B 的交叉反应性可以忽略不计（分别为 3.51%和 7.28%）。该 ic-ELISA 方法用于测定添加到米粉样品中的 Cry1C，平均回收率为 92.5%，变异系数（C.V.）小于 5.0%。本研究证明噬菌体展示文库为针对毒素靶标低成本、快速和连续产生特异性抗体片段提供了有价值的系统，并开发了一种简单的检测方法。我们的结果表明，Cry1C 特异性 scFv 可作为检测食品和农业样品中 Cry1C 的有用工具。
