title: Isolation of single chain variable fragment (scFv) specific for Cry1C toxin from human single fold scFv libraries.
abstract: 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.
authors.affiliation: 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.
authors.forename: Yun
authors.initials: Y
authors.lastname: Wang
authors.forename: Xiao
authors.initials: X
authors.lastname: Zhang
authors.forename: Cunzheng
authors.initials: C
authors.lastname: Zhang
authors.forename: Yuan
authors.initials: Y
authors.lastname: Liu
authors.forename: Xianjin
authors.initials: X
authors.lastname: Liu
mesh_terms: D000595:Amino Acid Sequence; D000083722:Bacillus thuringiensis Toxins; D001426:Bacterial Proteins / Q000032:analysis / Q000276:immunology*; D003429:Cross Reactions; D004731:Endotoxins / Q000032:analysis / Q000276:immunology*; D004797:Enzyme-Linked Immunosorbent Assay; D005506:Food Contamination; D006460:Hemolysin Proteins / Q000032:analysis / Q000276:immunology*; D006801:Humans; D008969:Molecular Sequence Data; D012275:Oryza / Q000737:chemistry; D019151:Peptide Library; D057127:Single-Chain Antibodies / Q000302:isolation & purification*
chemical_list: D000083722:Bacillus thuringiensis Toxins; D001426:Bacterial Proteins; D004731:Endotoxins; D006460:Hemolysin Proteins; D019151:Peptide Library; D057127:Single-Chain Antibodies; C030956:insecticidal crystal protein, Bacillus Thuringiensis