Established a new double antibodies sandwich enzyme-linked immunosorbent assay for detecting Bacillus thuringiensis (Bt) Cry1Ab toxin based single-chain variable fragments from a naïve mouse phage displayed library.
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 naïve 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 kDa. The purified scFv-G9 was used to develop a new DAS-ELISA for detecting Cry1Ab toxin, within minimum detection limit of 0.008 μg mL(-1), a working range 0.018-6.23 μg mL(-1), 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.
Key Lab of Food Quality and Safety of Jiangsu Province-State Key Laboratory Breeding Base, Key Laboratory of Control Technology and Standard for Agro-product Safety and Quality, Ministry of Agriculture, Institute of Food Quality Safety and Detection Research, Jiangsu Academy of Agricultural Sciences, 210014 Nanjing, PR China.
Xiao
X
Zhang
Key Lab of Food Quality and Safety of Jiangsu Province-State Key Laboratory Breeding Base, Key Laboratory of Control Technology and Standard for Agro-product Safety and Quality, Ministry of Agriculture, Institute of Food Quality Safety and Detection Research, Jiangsu Academy of Agricultural Sciences, 210014 Nanjing, PR China.
Chongxin
C
Xu
Key Lab of Food Quality and Safety of Jiangsu Province-State Key Laboratory Breeding Base, Key Laboratory of Control Technology and Standard for Agro-product Safety and Quality, Ministry of Agriculture, Institute of Food Quality Safety and Detection Research, Jiangsu Academy of Agricultural Sciences, 210014 Nanjing, PR China.
Cunzheng
C
Zhang
Key Lab of Food Quality and Safety of Jiangsu Province-State Key Laboratory Breeding Base, Key Laboratory of Control Technology and Standard for Agro-product Safety and Quality, Ministry of Agriculture, Institute of Food Quality Safety and Detection Research, Jiangsu Academy of Agricultural Sciences, 210014 Nanjing, PR China.
Yuan
Y
Liu
Key Lab of Food Quality and Safety of Jiangsu Province-State Key Laboratory Breeding Base, Key Laboratory of Control Technology and Standard for Agro-product Safety and Quality, Ministry of Agriculture, Institute of Food Quality Safety and Detection Research, Jiangsu Academy of Agricultural Sciences, 210014 Nanjing, PR China.
Yajing
Y
Xie
Key Lab of Food Quality and Safety of Jiangsu Province-State Key Laboratory Breeding Base, Key Laboratory of Control Technology and Standard for Agro-product Safety and Quality, Ministry of Agriculture, Institute of Food Quality Safety and Detection Research, Jiangsu Academy of Agricultural Sciences, 210014 Nanjing, PR China. Electronic address: jaasliu@jaas.ac.cn.
Xianjin
X
Liu
D000818:Animals; D001413:Bacillus thuringiensis / Q000235:genetics; D000083722:Bacillus thuringiensis Toxins; D001426:Bacterial Proteins / Q000032:analysis / Q000737:chemistry*; D003429:Cross Reactions; D004731:Endotoxins / Q000032:analysis / Q000737:chemistry*; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D005260:Female; D006460:Hemolysin Proteins / Q000032:analysis / Q000737:chemistry*; D051379:Mice; D008807:Mice, Inbred BALB C; D019151:Peptide Library; D030821:Plants, Genetically Modified / Q000737:chemistry*; D057127:Single-Chain Antibodies / Q000737:chemistry*
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
Cry1Ab toxin; Double antibodies sandwich immunoassay; Naïve mouse phage displayed library; ScFv