Site-saturation mutagenesis library construction and screening for specific broad-spectrum single-domain antibodies
Lingxia Jiao1,2, Yuan Liu1, Xiao Zhang1
1Key Laboratory of Food Quality and Safety of Jiangsu Province, Nanjing, 210014, China.
Abstract:
Potential ecological environmental and food safety risks of various Cry toxins of Bacillus thuringiensis (Bt) in transgenic food have received gradually increasing attention, which urged to establish an efficient and broad-spectrum detection technology for Cry toxins. Based on the single-domain antibody (sdAb) A8 against Bt Cry1Ab toxin screened from the humanized domain antibody library, the key amino acids of sdAb (A8) binding five kinds of Cry1 toxins were predicted using homology modeling and molecular docking technology, and the results showed that 105th asparagine, 106th arginine, 107th valine, and 114th arginine, respectively, located in heavy-chain complementarity-determining region 3 were common key amino acid sites. Subsequently, site-saturation cooperative mutagenesis of the four key sites was performed using overlap extension PCR, and multiple site-saturation mutagenesis sdAb library with the capacity of 1.2 × 105 colony-forming units (CFU) was successfully constructed. With alternating five Cry1 toxins as coating antigen, two generic sdAbs (2-C1, 2-C9) were screened out from the mutagenesis library, which could detect six kinds of Cry1 toxins at least. Through ELISA analysis, the binding activity of 2-C9 was significantly enhanced, and its OD values versus Cry1Aa, Cry1Ab, Cry1B, Cry1C, and Cry1E increased to 1.34, 1.53, 1.82, 2.39, and 2.7 times, respectively, compared with maternal antibody A8. The IC50 values of 2-C9 against Cry1Aa, Cry1Ab, Cry1B, and Cry1C were lower than that of A8, which showed that the affinity of 2-C9 against Cry1 toxins was enhanced. The results were beneficial to developing high-throughput and high-sensitive immune-detecting technology for Cry toxins.
Insights
Researchers engineered enhanced single-domain antibodies (sdAbs) for detecting Bacillus thuringiensis (Bt) Cry toxins in transgenic foods. This improved detection technology addresses food safety and environmental concerns by increasing sensitivity and broadening the spectrum of Cry toxins identified.
Area of Science:
- Biotechnology
- Immunology
- Food Safety
Background:
- Bacillus thuringiensis (Bt) Cry toxins in transgenic foods pose ecological, environmental, and food safety risks.
- Efficient and broad-spectrum detection technologies for Cry toxins are crucial for risk assessment and management.
Purpose of the Study:
- To develop an improved detection technology for Bacillus thuringiensis (Bt) Cry toxins.
- To engineer enhanced single-domain antibodies (sdAbs) with broader specificity and higher affinity for Cry toxins.
Main Methods:
- Homology modeling and molecular docking were used to predict key amino acid sites in sdAb A8 responsible for binding Cry1 toxins.
- Site-saturation mutagenesis was employed to construct a diverse sdAb library.
- Enzyme-linked immunosorbent assay (ELISA) was used to screen and characterize new sdAbs.
Main Results:
- Four key amino acid sites in the heavy-chain complementarity-determining region 3 were identified.
- A mutagenesis library of 1.2 × 10^5 colony-forming units (CFU) was constructed.
- Two generic sdAbs, 2-C1 and 2-C9, were screened, capable of detecting at least six Cry1 toxins.
- sdAb 2-C9 exhibited significantly enhanced binding activity and affinity against multiple Cry1 toxins compared to the original sdAb A8.
Conclusions:
- Engineered sdAbs, particularly 2-C9, demonstrate enhanced performance for Cry toxin detection.
- The developed technology offers a promising approach for high-throughput and highly sensitive immune-detection of Cry toxins.
- This advancement contributes to improved monitoring of transgenic food safety and environmental impact.
