Applied Microbiology and Biotechnology

, Volume 102, Issue 7, pp 3363–3374 | Cite as

Screening for single-chain variable fragment antibodies against multiple Cry1 toxins from an immunized mouse phage display antibody library

  • Sa Dong
  • Zongyi Bo
  • Cunzheng Zhang
  • Jianguo Feng
  • Xianjin Liu
Methods and protocols


Single-chain variable fragment (scFv) is a kind of antibody that possess only one chain of the complete antibody while maintaining the antigen-specific binding abilities and can be expressed in prokaryotic system. In this study, scFvs against Cry1 toxins were screened out from an immunized mouse phage displayed antibody library, which was successfully constructed with capacity of 6.25 × 107 CFU/mL. Using the mixed and alternative antigen coating strategy and after four rounds of affinity screening, seven positive phage-scFvs against Cry1 toxins were selected and characterized. Among them, clone scFv-3H9 (MG214869) showing relative stable and high binding abilities to six Cry1 toxins was selected for expression and purification. SDS-PAGE indicated that the scFv-3H9 fragments approximately 27 kDa were successfully expressed in Escherichia coli HB2151 strain. The purified scFv-3H9 was used to establish the double antibody sandwich enzyme-linked immunosorbent assay method (DAS-ELISA) for detecting six Cry1 toxins, of which the lowest detectable limits (LOD) and the lowest quantitative limits (LOQ) were 3.14–11.07 and 8.22–39.44 ng mL−1, respectively, with the correlation coefficient higher than 0.997. The average recoveries of Cry1 toxins from spiked rice leaf samples were ranged from 84 to 95%, with coefficient of variation (CV) less than 8.2%, showing good accuracy for the multi-residue determination of six Cry1 toxins in agricultural samples. This research suggested that the constructed phage display antibody library based on the animal which was immunized with the mixture of several antigens under the same category can be used for the quick and effective screening of generic antibodies.


Cry1 toxins ScFv antibody Phage displayed antibody library Generic antibody DAS-ELISA 



This research was supported by the General Financial Grant from the China Postdoctoral Science Foundation (No. 2017M621674), the Special Fund for Agro-scientific Research in the Public Interest (201303088), the Natural Science Foundation of Jiangsu Province (BK20131333), and the Basic Research Project (Natural Science Foundation for Young Scholars) of Jiangsu Province, China (Grant No. BK20170489).

Compliance with ethical standards

All involved animal experiments in this research were in compliance with ethical standards.

Conflict of interest

The authors declare that they have no conflict of interest.


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Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2018

Authors and Affiliations

  1. 1.College of Horticulture and Plant ProtectionYangzhou UniversityYangzhouPeople’s Republic of China
  2. 2.Laboratory for Food Quality and Safety-State Key Laboratory Cultivation Base of Ministry of Science and Technology, Key Laboratory of Control Technology and Standard for Agro-product Safety and Quality, Ministry of AgricultureJiangsu Academy of Agricultural SciencesNanjingPeople’s Republic of China
  3. 3.College of Veterinary MedicineNanjing Agricultural UniversityNanjingPeople’s Republic of China

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