title: Production of a soluble single-chain variable fragment antibody against okadaic acid and exploration of its specific binding.
abstract: Okadaic acid is a lipophilic marine algal toxin commonly responsible for diarrhetic shellfish poisoning (DSP). Outbreaks of DSP have been increasing and are of worldwide public health concern; therefore, there is a growing demand for more rapid, reliable, and economical analytical methods for the detection of this toxin. In this study, anti-okadaic acid single-chain variable fragment (scFv) genes were prepared by cloning heavy and light chain genes from hybridoma cells, followed by fusion of the chains via a linker peptide. An scFv-pLIP6/GN recombinant plasmid was constructed and transformed into Escherichia coli for expression, and the target scFv was identified with IC-CLEIA (chemiluminescent enzyme immunoassay). The IC15 was 0.012 ± 0.02 μg/L, and the IC50 was 0.25 ± 0.03 μg/L. The three-dimensional structure of the scFv was simulated with computer modeling, and okadaic acid was docked to the scFv model to obtain a putative structure of the binding complex. Two predicted critical amino acids, Ser32 and Thr187, were then mutated to verify this theoretical model. Both mutants exhibited significant loss of binding activity. These results help us to understand this specific scFv-antigen binding mechanism and provide guidance for affinity maturation of the antibody in vitro. The high-affinity scFv developed here also has potential for okadaic acid toxin detection.
authors.affiliation: Key Laboratory of Food Nutrition and Safety, Ministry of Education of China, Tianjin University of Science and Technology, Tianjin 300457, China; Hebei North University, Zhangjiakou 075000, China.
authors.forename: Kuo
authors.initials: K
authors.lastname: He
authors.affiliation: Hebei North University, Zhangjiakou 075000, China.
authors.forename: Xiuyuan
authors.initials: X
authors.lastname: Zhang
authors.affiliation: Hebei North University, Zhangjiakou 075000, China.
authors.forename: Lixia
authors.initials: L
authors.lastname: Wang
authors.affiliation: Key Laboratory of Food Nutrition and Safety, Ministry of Education of China, Tianjin University of Science and Technology, Tianjin 300457, China.
authors.forename: Xinjun
authors.initials: X
authors.lastname: Du
authors.affiliation: Hebei North University, Zhangjiakou 075000, China. Electronic address: weidongss1971@sina.com.
authors.forename: Dong
authors.initials: D
authors.lastname: Wei
mesh_terms: D000918:Antibody Specificity; D001666:Binding Sites, Antibody; D007124:Immunoenzyme Techniques; D049449:Luminescence; D019319:Okadaic Acid / Q000032:analysis / Q000737:chemistry / Q000276:immunology*; D057127:Single-Chain Antibodies / Q000737:chemistry* / Q000235:genetics / Q000276:immunology*; D012995:Solubility
chemical_list: D057127:Single-Chain Antibodies; D019319:Okadaic Acid
keywords: Binding complex; CLEIA; Okadaic acid; Single-chain variable fragment