Production of a soluble single-chain variable fragment antibody against okadaic acid and exploration of its specific binding

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• 作者：Kuo He^a ; ^b ; Xiuyuan Zhang^b ; Lixia Wang^b ; Xinjun Du^a ; Dong Wei^b ; ^{weidongss1971@sina.com" class="auth_mail" title="E-mail the corresponding author}
• 关键词：Okadaic acid ; CLEIA ; Single-chain variable fragment ; Binding complex
• 刊名：Analytical Biochemistry
• 出版年：2016
• 出版时间：15 June 2016
• 年：2016
• 卷：503
• 期：Complete
• 页码：21-27
• 全文大小：1228 K

文摘

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.