Production of recombinant ScFv antibodies against methamidophos from a phage-display library of a hyperimmunized...

A recombinant phage display library was generated using splenocyte mRNA isolated from a Balb/c mouse hyperimmunized with a hapten conjugate that mimicked the structure of methamidophos, one of the most acutely toxic organophosphate pesticides. Three recombinant single-chain variable fragment (scFv) antibodies with the highest specificity for methamidophos, termed 28D4, 29D0, and 36B2, were produced via a stringent selection protocol. In a competitive enzyme-linked immunosorbent assay, 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 below 0.1% except for acephate (O,S-dimethyl acetylphosphoramidothioate). Nucleotide and deduced amino acid sequences indicated that the respective heavy chains and light chains of the three scFvs were involved in the distinctive VDJ segment rearrangements associated with somatic hypermutations during the process of several immunizations with higher dosages of immunogen. Taken together, these data constitute the first detailed description of an immunoassay that utilizes scFvs against the methamidophos, an analyte with a simple structure and low molecular mass (141 Da).