Production of Recombinant ScFv Antibodies against Methamidophos from a Phage-Display Library of a Hyperimmunized Mouse

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• 作者：Tiejun Li ; Qi Zhang ; Yan Liu ; Defeng Chen ; Baishi Hu ; Diane A. Blake ; Fengquan Liu
• 刊名：Journal of Agricultural and Food Chemistry
• 出版年：2006
• 出版时间：November 29, 2006
• 年：2006
• 卷：54
• 期：24
• 页码：9085 - 9091
• 全文大小：71K
• 年卷期：v.54,no.24(November 29, 2006)
• ISSN：1520-5118

文摘

A recombinant phage display library was generated using splenocyte mRNA isolated from a Balb/cmouse hyperimmunized with a hapten conjugate that mimicked the structure of methamidophos,one of the most acutely toxic organophosphate pesticides. Three recombinant single-chain variablefragment (scFv) antibodies with the highest specificity for methamidophos, termed 28D4, 29D0, and36B2, were produced via a stringent selection protocol. In a competitive enzyme-linked immunosorbentassay, the IC₅₀ values for 28D4, 29D0, and 36B2 were 46.25, 35.39, and 17.99 ng/mL, respectively.The cross-reactivity of the three scFv antibodies with other organophosphate pesticides was below0.1% except for acephate (O,S-dimethyl acetylphosphoramidothioate). Nucleotide and deduced aminoacid sequences indicated that the respective heavy chains and light chains of the three scFvs wereinvolved in the distinctive VDJ segment rearrangements associated with somatic hypermutations duringthe process of several immunizations with higher dosages of immunogen. Taken together, thesedata constitute the first detailed description of an immunoassay that utilizes scFvs against themethamidophos, an analyte with a simple structure and low molecular mass (141 Da).