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Selection and application of broad-specificity human domain antibody for simultaneous detection of Bt Cry toxins
Chongxin Xu1, Xiao Zhang1, Xiaoqin Liu2
1Key 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.
Abstract:
Bt Cry toxin is a kind of bio-toxins that used for genetically modified crops (GMC) transformation widely. In this study, total 15 positive clones could bind the Bt Cry toxins which isolated from a human domain antibody library by 5 rounds affinity selection. According to analyzing of PCR amplification and enzyme-linked immunosorbent assay (ELISA), the most positive phage domain antibody (named F5) gene was cloned into the pET26b vector and expressed in E. coli BL21. The purified antibody was used to develop an indirect competitive ELISA (IC-ELISA) for Cry1Ab, Cry1Ac, Cry1B, Cry1C and Cry1F toxins, respectively. The working range of detection for standard curves in IC-ELISA were 0.258-1.407 μg/mL, the medium inhibition concentration (IC50) were 0.727-0.892 μg/mL and detection limit (IC10) were 0.029-0.074 μg/mL for those Bt Cry toxins. The affinity of F5 domain antibody with Cry1Ab, Cry1Ac, Cry1B, Cry1C and Cry1F toxins were 1.21-5.94 × 10(7) M(-1). The average recoveries of the 5 kinds of Bt Cry toxins from spiked wheat samples were ranged from 81.2%-100.8% with a CV at 2.5%-9.4%. The results showed that we successfully obtained the broad-specificity human domain antibody for simultaneous detection of Bt Cry toxins in agricultural product samples.
Insights
Researchers developed a human domain antibody for detecting multiple Bt Cry toxins in genetically modified crops. This antibody enables simultaneous detection of these bio-toxins in agricultural products.
Area of Science:
- Biotechnology
- Immunology
- Analytical Chemistry
Background:
- Bacillus thuringiensis (Bt) Cry toxins are widely used in genetically modified crops (GMC).
- Accurate detection of Bt Cry toxins is crucial for agricultural product safety and regulation.
- Existing detection methods may lack specificity or efficiency for multiple toxin types.
Purpose of the Study:
- To isolate and characterize a human domain antibody with broad specificity for Bt Cry toxins.
- To develop a sensitive and reliable immunoassay for simultaneous detection of various Bt Cry toxins.
- To validate the antibody's performance in detecting Bt Cry toxins in agricultural samples.
Main Methods:
- Affinity selection using a human domain antibody library against Bt Cry toxins.
- Gene cloning, expression, and purification of the selected antibody (F5).
- Development and validation of an indirect competitive ELISA (IC-ELISA) for toxin detection.
Main Results:
- A human domain antibody (F5) was successfully generated, showing binding affinity to multiple Bt Cry toxins (Cry1Ab, Cry1Ac, Cry1B, Cry1C, Cry1F).
- The IC-ELISA demonstrated a detection range of 0.258-1.407 μg/mL, with IC50 values between 0.727-0.892 μg/mL and detection limits (IC10) of 0.029-0.074 μg/mL.
- The antibody achieved high recovery rates (81.2%-100.8%) with low coefficients of variation (2.5%-9.4%) in spiked wheat samples.
Conclusions:
- A broad-specificity human domain antibody capable of simultaneously detecting multiple Bt Cry toxins was successfully developed.
- The developed IC-ELISA method is sensitive, specific, and suitable for the detection of Bt Cry toxins in agricultural products.
- This antibody provides a valuable tool for ensuring the safety and quality of agricultural products containing Bt Cry toxins.
