Screening and detection of Cry2Aa-binding specific single chain antibody fragments (scFv) from a humanized phage display library by magnetic beads

WU Ai-hua;WANG Yun;LIU Yuan;HU Xiao-dan;LIU Xian-jin;Institute of Food Quality Safety and Detection Research,Jiangsu Academy of Agricultural Sciences/Key Laboratory of Food Quality and Jiangsu Province-State Key Laboratory Breeding Base/Key Laboratory of Control Technology and Standard for Agro-product Safety and Quality,Ministry of Agriculture;College of Horticulture,Jinling Institute of Technology;

Cry2Aa toxin;;specific single chain antibody fragments (scFv);;magnetic bead;;indirect-competitive ELISA

To develop a rapid detection method of transgenic component in agricultural products,specific single chain antibody fragments (sc Fv) against Cry2Aa toxin were isolated from humanized phage antibody library (Tomlinson I),by optimizing the magnetic beads and liquid spanning strategy.After 4 rounds of"amplification-adsorption-elution",single colonies were randomly selected from counting plate of the last round for identification.After DNA sequencing,the positive clones with better binding activity were selected to develop an indirect competitive ELISA,and the detection range of the ELISA was determined.The relative yield of the fourth round of screening was 4.07 × 106 times higher than that of the first round.Totally 8 positive clones were identified by indirect competitive ELISA,which recognized Cry2Aa specifically and contained the complete gene fragment.The positive clone D5 showed minimum detection limit of 6.632 ng/ml and linear range of detection of approximately0.016-2.881 μg/ml ability than others was employed to develop an indirect for Cry2Aa detection by the ELISA method.This method developed in this study has good specificity for Cry2Aa and reproducibility between the plates.