Production and characterization of a single-chain variable fragment antibody from a site-saturation mutagenesis library derived from the anti-Cry1A monoclonal antibody

Abstract

There are plenty of applications of Cry1A toxins (Cry1Aa, Cry1Ab, Cry1Ac) in genetically modified crops, and it is necessary to establish corresponding detection methods. In this study, a single-chain variable fragment (scFv) with high affinities to Cry1A toxins was produced. First, the variable regions of heavy (V_H) and light chain (V_L) were amplified from hybridoma cell 5B5 which secrete anti-Cry1A monoclonal antibody (mAb) and then spliced into scFv-5B5 by overlap extension polymerase chain reaction (SOE-PCR). Subsequently, site-saturation mutagenesis was performed after homology modeling and molecular docking, which showed that asparagine³⁵, phenylalanine³⁶, isoleucine¹⁰⁴, tyrosine¹⁰⁵, and serine¹⁹⁶, respectively, located in V_H complementarity-determining region (CDR1 and CDR3) and V_L framework region (FR3) were key amino acid sites. Then, the mutagenesis scFv library (1.35 × 10⁵ CFU/mL) was constructed and a mutant scFv-2G12 with equilibrium dissociation constant (K_D) value of 9.819 × 10^-9 M against Cry1Ab toxin, which was lower than scFv-5B5 (2.025 × 10^-8 M) was obtained by biopanning. Then, enzyme-linked immunosorbent assay (ELISA) was established with limit of detection (LOD) and limit of quantitation (LOQ) of 4.6-9.2 and 11.1-17.1 ng mL^-1 respectively for scFv-2G12, which were lower than scFv-5B5 (12.4-22.0 and 23.6-39.7 ng mL^-1). Results indicated the promising prospect of scFv-2G12 used for the detection of Cry1A toxins.

Keywords: Cry1A toxins; ScFv antibody; Site-saturation mutagenesis.