title: Establishment of a sensitive time-resolved fluoroimmunoassay for detection of Bacillus thuringiensis Cry1Ie toxin based nanobody from a phage display library.
abstract: Cry1Ie toxin was an insect-resistant protein used in genetically modified crops (GMC). In this study, a large human VH gene nanobodies phage displayed library was employed to select anti-Cry1Ie toxin antibody by affinity panning. After 5 rounds of panning, total 12 positive monoclonal phage particles were obtained. One of the identified positive phage nanobody was expressed in E.coli BL21 and the purified protein was indicated as a molecular mass of approximately 20 kDa by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Then a sensitive indirect competitive time-resolved fluoroimmunoassay (IC-TRFIA) was established for detection of Cry1Ie toxin by the purified protein. The working range of detection for Cry1Ie toxin standards in the IC-TRFIA were 0.08-6.44 ng mL-1and the medium inhibition of control (IC50) was 0.73 ng mL-1. It showed a weak cross-reactivity with Cry1Ab toxin (at 5.6%), but did not recognize Cry1B, Cry1C, Cry1F, and Cry2A toxins (were <0.1%). The average recoveries of Cry1Ie toxin from respectively spiked in rice, corn and soil samples were in the range of 83.5%-96.6% and with a coefficient of variation (CV) among 2.0%-8.6%. These results showed the IC-TRFIA was promising for detection of Cry1Ie toxin in agricultural and environmental samples.
authors.affiliation: Key Laboratory of Food Quality and Safety of Jiangsu/Province-State Key Laboratory Breeding Base, Institute of Food Quality Safety and Detection, Jiangsu Academy of Agricultural Sciences, Nanjing 210014, China.
authors.forename: Chongxin
authors.initials: C
authors.lastname: Xu
authors.affiliation: Huaihua Vocational and Technical College, Huaihua 418007, China.
authors.forename: Xiaoqin
authors.initials: X
authors.lastname: Liu
authors.affiliation: Key Laboratory of Food Quality and Safety of Jiangsu/Province-State Key Laboratory Breeding Base, Institute of Food Quality Safety and Detection, Jiangsu Academy of Agricultural Sciences, Nanjing 210014, China.
authors.forename: Cunzheng
authors.initials: C
authors.lastname: Zhang
authors.affiliation: Key Laboratory of Food Quality and Safety of Jiangsu/Province-State Key Laboratory Breeding Base, Institute of Food Quality Safety and Detection, Jiangsu Academy of Agricultural Sciences, Nanjing 210014, China.
authors.forename: Xiao
authors.initials: X
authors.lastname: Zhang
authors.affiliation: Key Laboratory of Food Quality and Safety of Jiangsu/Province-State Key Laboratory Breeding Base, Institute of Food Quality Safety and Detection, Jiangsu Academy of Agricultural Sciences, Nanjing 210014, China.
authors.forename: Jianfeng
authors.initials: J
authors.lastname: Zhong
authors.affiliation: Key Laboratory of Food Quality and Safety of Jiangsu/Province-State Key Laboratory Breeding Base, Institute of Food Quality Safety and Detection, Jiangsu Academy of Agricultural Sciences, Nanjing 210014, China.
authors.forename: Yuan
authors.initials: Y
authors.lastname: Liu
authors.affiliation: Key Laboratory of Food Quality and Safety of Jiangsu/Province-State Key Laboratory Breeding Base, Institute of Food Quality Safety and Detection, Jiangsu Academy of Agricultural Sciences, Nanjing 210014, China.
authors.forename: Xiaodan
authors.initials: X
authors.lastname: Hu
authors.affiliation: Key Laboratory of Food Quality and Safety of Jiangsu/Province-State Key Laboratory Breeding Base, Institute of Food Quality Safety and Detection, Jiangsu Academy of Agricultural Sciences, Nanjing 210014, China.
authors.forename: Manman
authors.initials: M
authors.lastname: Lin
authors.affiliation: Key Laboratory of Food Quality and Safety of Jiangsu/Province-State Key Laboratory Breeding Base, Institute of Food Quality Safety and Detection, Jiangsu Academy of Agricultural Sciences, Nanjing 210014, China. Electronic address: hhxyxcx@163.com.
authors.forename: Xianjin
authors.initials: X
authors.lastname: Liu
mesh_terms: D001413:Bacillus thuringiensis / Q000737:chemistry*; D000083722:Bacillus thuringiensis Toxins; D001426:Bacterial Proteins / Q000032:analysis*; D004731:Endotoxins / Q000032:analysis*; D015200:Fluoroimmunoassay / Q000379:methods; D005504:Food Analysis / Q000379:methods*; D006460:Hemolysin Proteins / Q000032:analysis*; D006801:Humans; D012275:Oryza / Q000737:chemistry*; D019151:Peptide Library; D061905:Single-Domain Antibodies / Q000737:chemistry*
chemical_list: D000083722:Bacillus thuringiensis Toxins; D001426:Bacterial Proteins; D004731:Endotoxins; D006460:Hemolysin Proteins; D019151:Peptide Library; D061905:Single-Domain Antibodies; C030956:insecticidal crystal protein, Bacillus Thuringiensis
keywords: Cry1Ie toxin; Nanobody; Phage display library; Time-resolved fluoroimmunoassay (TRFIA)