Construction of a single-chain variable fragment antibody against daidzin and its potential use in an enzyme-linked immunosorbent assay

Daidzin is one of the major isoflavonoids found in legumes. Several studies has reported estrogenic effects of this class of compounds. To develop an immunoassay to determine the concentration of daidzin in plant samples, a single-chain variable fragment antibody (scFv) against daidzin was constructed. The variable heavy and light chain genes were cloned from cDNA of hybridoma cell line. Then they were assembled together by splicing by overlap extension PCR (SOE-PCR) using specific primers designed to have flexible peptide (Gly₍₄₎Ser)₍₃₎ as linker. The constructed scFv gene was ligated into the pET28a expression vector and transformed into E. coli BL21 (DE3). The expressed scFv containing His6-tag at its N-termini was purified by immobilized metal ion affinity chromatography (IMAC). The purified scFv was characterized and applied to use in enzyme-linked immunosorbent assay (ELISA).