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

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.

Site-saturation mutagenesis library construction and screening for specific broad-spectrum single-domain antibodies against multiple Cry1 toxins.

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.

Alteration of a Cry1A Shared Binding Site in a Cry1Ab-Selected Colony of Ostrinia furnacalis.

The Asian corn borer, Ostrinia furnacalis (Guenée, 1854), is a highly damaging pest in Asia and the Pacific islands, and larvae feed mainly from corn crops. To determine the suitability of Bt-corn technology for the future control of this pest, understanding the potential to develop resistance to Cry1Ab and the basis of cross-resistance to other Cry1 proteins is of great interest. Here, we have explored the binding of Cry1A proteins to brush border membrane vesicles from two O. furnacalis colonies, one susceptible (ACB-BtS) and one laboratory-selected with Cry1Ab (ACB-AbR). The insects developed resistance to Cry1Ab and showed cross-resistance to Cry1Aa, Cry1Ac, and Cry1F. Binding assays with radiolabeled Cry1Ab and brush border membrane vesicles from susceptible insects showed that Cry1A proteins shared binding sites, though the results were not conclusive for Cry1F. The results were confirmed using radiolabeled Cry1Aa. The resistant insects showed a reduction of the specific binding of both Cry1Ab and Cry1Aa, suggesting that part of the binding sites were lost or altered. Competition binding assays showed full competition between Cry1Ab and Cry1Aa proteins in the susceptible colony but only partial competition in resistant insects, confirming the alteration of some, but not all, binding sites for these two proteins. The binding site model for Cry1A proteins in O. furnacalis is in agreement with the occurrence of multiple membrane receptors for these proteins.