Establishment of monoclonal antibody and scFv immuno-based assay for Cry2Aa toxin in spiked grain samples

Cheng Shen; Jia Hao; Yihang Li; Jiafeng Jin; Meng Meng; Xiao Zhang; Manman Lin; Chongxin Xu; Qin Zhu; Yajing Xie; Johnson Lin; Yuan Liu; Xianjin Liu

doi:10.1016/j.ab.2023.115270

Establishment of monoclonal antibody and scFv immuno-based assay for Cry2Aa toxin in spiked grain samples

Anal Biochem. 2023 Sep 15:677:115270. doi: 10.1016/j.ab.2023.115270. Epub 2023 Jul 31.

Authors

Cheng Shen¹, Jia Hao¹, Yihang Li¹, Jiafeng Jin², Meng Meng², Xiao Zhang², Manman Lin², Chongxin Xu², Qin Zhu², Yajing Xie², Johnson Lin³, Yuan Liu⁴, Xianjin Liu⁵

Affiliations

¹ Key Laboratory of Food Quality and Safety of Jiangsu Province, Nanjing, 210014, China; College of Plant Protection, Nanjing Agricultural University, Nanjing, 210095, China.
² Key Laboratory of Food Quality and Safety of Jiangsu Province, Nanjing, 210014, China.
³ School of Life Sciences, Discipline of Microbiology, College of Agriculture, Engineering and Science, University of KwaZulu-Natal, Westville Campus, Private Bag X54001, Durban, 4000, South Africa.
⁴ Key Laboratory of Food Quality and Safety of Jiangsu Province, Nanjing, 210014, China. Electronic address: liuyuan@jaas.ac.cn.
⁵ Key Laboratory of Food Quality and Safety of Jiangsu Province, Nanjing, 210014, China; College of Plant Protection, Nanjing Agricultural University, Nanjing, 210095, China. Electronic address: jaasliu@jaas.ac.cn.

PMID: 37531991
DOI: 10.1016/j.ab.2023.115270

Abstract

Bacillus thuringiensis (Bt) Cry toxins have been widely used in the development of genetically modified organisms (GMOs) for pest control. This work aimed to establish more cost effective methods for used Cry2Aa toxins. Three immunoassay methods (IC-ELISA, DAS-ELISA, and CLEIA) were successfully developed in this work. The mAb was used as the detecting antibody, for the IC-ELISA, the range of IC₂₀ to IC₈₀ was 1.11 μg/mL - 60.70 μg/mL, and an IC₅₀ of 10.65 μg/mL. For the DAS-ELISA, the limit of detection (LOD) and limit of quantitation (LOQ) were 10.76 ng/mL and 20.70 ng/mL, respectively. For the CLEIA, the LOD and LOQ were 6.17 ng/mL and 7.40 ng/mL, respectively. The scFv-based detections were the most sensitive for detecting Cry2Aa. The LOD and LOQ for the DAS-ELISA were 118.75 ng/mL and 633.48 ng/mL, respectively. The LOD and LOQ for the CLEIA, read as 37.47 ng/mL and 70.23 ng/mL, respectively. The fact that Cry2Aa toxin was recovered in spiked grain samples further demonstrated that the approaches might be used to identify field samples. These methods provided good sensitivity, stability, and applicability for detecting Cry2Aa toxin, promising ultrasensitive monitoring and references for Cry toxins risk assessment.

Keywords: CLIEA; Cry2Aa; DAS-ELISA; IC-ELISA; MAb; ScFv.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

Antibodies, Monoclonal*
Bacillus thuringiensis*
Bacterial Proteins / analysis
Endotoxins / analysis
Enzyme-Linked Immunosorbent Assay / methods
Hemolysin Proteins

Substances

Antibodies, Monoclonal
Bacterial Proteins
Endotoxins
Hemolysin Proteins