Construction of an Immunized Rabbit Phage Display Library for Selecting High Activity against Bacillus thuringiensis Cry1F Toxin Single-Chain Antibodies

In the present study, a Cry1F-immunized rabbit phage display library (6.96×108 23 ) was 24 constructed for selecting high activity of anti-Cry1F toxin single-chain antibody (scFv) by bio-panning. 25 Total 16 positive monoclonal phage scFvs were obtained after 4 rounds panning which identified by 26 enzyme-linked immunoassay (ELISA), PCR and DNA sequencing. The most positive phage scFv 27 (named RF4) was expressed in E. coli.HB2151 and the purified soluble protein approximate 30 kDa 28 with sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). An indirect-competitive 29 ELISA (IC-ELISA) was established base on purified soluble RF4-scFv for Cry1F toxin and indicated the 50 % inhibition of control (IC50) was 11.56 ng/mL, the detection limit (IC10 30 ) was 0.18 ng/mL, and 31 showed a weakly cross-reactivity (CR) for Cry1Ab (2.8 %), Cry1Ac (1.3 %), Cry1B, Cry1C, Cry1Ie 32 and Cry2A (less than 0.1 %). It had a good accuracy, stability and repeatability that Cry1F toxin spiked 33 in rice, wheat, corn and soil samples by IC-ELISA, the recoveries were in the range of 80.2 %~99.6 % 34 and the coefficient of variation (CVs) were among 2.5 %~10.0 %. These results showed the IC-ELISA 35 based on scFv from the immunized rabbit phage display library was promising for specific-detecting 36 Cry1F toxin in agro-products and environmental samples.