Construction and Characterization of a Single-Chain Variable Fragment (scFv) for the Detection of Cry1Ab and Cry1Ac Toxins from the Anti-Cry1Ab Monoclonal Antibody

In the previous study, monoclonal antibody 2C12 which could specifically recognize Cry1Ab toxin and has no cross-reaction with Cry1Ac toxin was prepared by using the synthetic polypeptide T4 as immunogen. In this study, 2C12 was used as the gene source, and the single chain antibody scFv-2C12 was successfully constructed and expressed. Indirect ELISA result showed that scFv-2C12 could recognize both Cry1Ab and Cry1Ac toxins. In order to clarify the recognition mechanism of scFv-2C12 with Cry1Ab and Cry1Ac toxins, the interaction models of scFv-2C12 with Cry1Ab and Cry1Ac toxins were built and analyzed using homology modeling and molecular docking techniques. The results showed that scFv-2C12 was able to penetrate deep into Cry1Ac toxin and recognize hidden epitopes that could not recognized by conventional monoclonal antibody 2C12. The number of hydrogen bonds formed between scFv-2C12 and Cry1Ab/Ac was nine and eight, separately. The hydrophobic interactions formed between scFv-2C12 and Cry1Ab/Ac were both eight. Subsequently, based on scFv-2C12, double-antibody sandwich ELISA (DAS-ELISA) methods were developed to detect Cry1Ab and Cry1Ac toxins. The results showed that the minimum limits of detection and quantification (LOD and LOQ) for Cry1Ab were 5.014 and 20.45 ng center dot mL(-1), and 7.409 and 22.01 ng center dot mL(-1) for Cry1Ac, respectively. This study provides a basic material for the establishment of detection method for Cry1Ab and Cry1Ac toxins, and provides a new idea for the preparation of broad-spectrum antibodies.

Automated summary

In this study, a monoclonal antibody 2C12, which specifically recognizes Cry1Ab toxin without cross-reaction with Cry1Ac toxin, was used as the gene source to construct and express the single chain antibody scFv-2C12. The interaction models between scFv-2C12 and Cry1Ab/Cry1Ac were built and analyzed, revealing that scFv-2C12 could recognize hidden epitopes in Cry1Ac toxin. Furthermore, a double-antibody sandwich ELISA method was developed based on scFv-2C12 for the detection of Cry1Ab and Cry1Ac toxins.