Screening and Molecular Evolution of a Single Chain Variable Fragment Antibody (scFv) against Citreoviridin Toxin

Citreoviridin (CIT), a small food-borne mycotoxin produced by Penicillium citreonigrum, is generally distributed in various cereal grains and farm crop products around the world and has caused cytotoxicity as an uncompetitive inhibitor of ATP hydrolysis. A high affinity single chain variable fragment (scFv) antibody that can detect the citreoviridin in samples is still not available; therefore, it is very urgent to prepare an antibody for CIT detection and therapy. In this study, an amplified and assembled scFv from hybridoma was used to construct the mutant phage library by error-prone PCR, generating a 2 × 10⁸ capacity mutated phage display library. After six rounds of biopanning, the selected scFv-5A10 displayed higher affinity and specificity to CIT antigen, with an increased affinity of 13.25-fold (K_aff = 5.7 × 10⁹ L/mol) compared to that of the original wild-type scFv. Two critical amino acids (P100 and T151) distributed in H-CDR3 and L-FR regions that were responsible for scFv-5A10 to CIT were found and verified by oligonucleotide-directed mutagenesis, and the resulting three mutants except for the mutant (P100K) lost binding activity significantly against CIT, as predicated. Indirect competitive ELISA (ic-ELISA) indicated that the linear range to detect CIT was 25-562 ng/mL with IC₅₀ at 120 ng/mL. The limit of detection was 14.7 ng/mL, and the recovery average was (90.612 ± 3.889)%. Hence, the expressed and purified anti-CIT MBP-linker-scFv can be used to detect CIT in corn and related samples.