Construction of an Immunized Rabbit Phage Display Library for Selecting High Activity against Bacillus thuringiensis Cryl F Toxin Single-Chain Antibodies

In the present study, a Cry1F-immunized rabbit phage display library (6.96 X 10(8) cfu/mL) was constructed for selecting high activity of anti-CrylF toxin single-chain antibody (a single-chain variable fragment, scFv) by biopanning. A total of 16 positive monoclonal phage scFv's were obtained after 4 rounds of panning, which were identified by enzyme-linked immunosorbent assay (ELISA), polymerized chain reaction, and DNA sequencing. The most positive phage scFv (named RF4) was expressed in Escherichia coli HB2151, and a soluble protein of approximately 30 kDa was purified with sodium dodecyl sulfate polyacrylamide gel electrophoresis. An indirect competitive ELISA (IC-ELISA) was established on the basis of purified soluble RF4-scFv for CrylF toxin. It indicated the 50% inhibition of the control (IC50) was 11.56 ng/mL and the detection limit (IC10) was 0.18 ng/mL and showed weak cross-reactivities for CrylAb (2.8%), CrylAc (1.3%), and Cry1B, Cry1C, Crylle, and Cry2A (less than 0.1%). It was found that IC-ELISA detected Cry1F toxin spiked in rice, wheat, corn, and soil samples with good accuracy, stability, and repeatability. The recoveries were in the range of 80.2-99.6%, and the coefficients of variation were in the range of 2.5-10.0%. These results showed that IC-ELISA based on scFv from the immunized rabbit phage display library was promising for specific detection of Cry1F toxin in agroproducts and environmental samples.