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Establishment of a sandwich enzyme-linked immunosorbent assay for specific detection of Bacillus thuringiensis (Bt) Cry1Ab toxin utilizing a monoclonal antibody produced with a novel hapten designed with molecular model

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Abstract

Cry1Ab toxin is commonly expressed in genetically modified crops in order to control chewing pests. At present, the detection method with enzyme-linked immunosorbent assay (ELISA) based on monoclonal antibody cannot specifically detect Cry1Ab toxin for Cry1Ab’s amino acid sequence and spatial structure are highly similar to Cry1Ac toxin. In this study, based on molecular design, a novel hapten polypeptide was synthesized and conjugated to keyhole limpet hemocyanin (KLH). Then, through animal immunization with this antigen, a monoclonal antibody named 2C12, showing high affinity to Cry1Ab and having no cross reaction with Cry1Ac, was produced. The equilibrium dissociation constant (K D) value of Cry1Ab toxin with MAb 2C12 was 1.947 × 10−8 M. Based on this specific monoclonal antibody, a sandwich enzyme-linked immunosorbent assay (DAS-ELISA) was developed for the specific determination of Cry1Ab toxin and the LOD and LOQ values were determined as 0.47 ± 0.11 and 2.43 ± 0.19 ng mL−1, respectively. The average recoveries of Cry1Ab from spiked rice leaf and rice flour samples ranged from 75 to 115%, with coefficient of variation (CV) less than 8.6% within the quantitation range (2.5–100 ng mL−1), showing good accuracy for the quantitative detection of Cry1Ab toxin in agricultural samples. In conclusion, this study provides a new approach for the production of high specific antibody and the newly developed DAS-ELISA is a useful method for Cry1Ab monitoring in agriculture products.

Establishment of a DAS-ELISA for the specific detecting of Bacillus thuringiensis (Bt) Cry1Ab toxin

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Acknowledgements

This work was supported by the National Natural Science Foundation of China (General Program) (No. 31371778), the Special Fund for Agro-scientific Research in the Public Interest (201303088), the Jiangsu Province Natural Science Foundation (BK20131333), the Key Technology R&D Program of Jiangsu Province (No. BE2014722), the National Natural Science Foundation of China (No. 31301703), and the Independent Innovation Project of Jiangsu Province (CX (14) 5068).

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  1. 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, Jiangsu Academy of Agricultural Sciences, Nanjing, Jiangsu, 210014, China

    Sa Dong, Xiao Zhang, Yuan Liu, Cunzheng Zhang, Yajing Xie, Jianfeng Zhong, Chongxin Xu & Xianjin Liu

  2. College of Plant Protection, Nanjing Agricultural University, Nanjing, Jiangsu, 210095, China

    Sa Dong & Xianjin Liu

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Dong, S., Zhang, X., Liu, Y. et al. Establishment of a sandwich enzyme-linked immunosorbent assay for specific detection of Bacillus thuringiensis (Bt) Cry1Ab toxin utilizing a monoclonal antibody produced with a novel hapten designed with molecular model. Anal Bioanal Chem 409, 1985–1994 (2017). https://doi.org/10.1007/s00216-016-0146-0

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