Single-chain variable fragment antibodies targeting the antileukemia agent harringtonine for enzyme-linked immunosorbent assay development

In this study, we generated a single-chain variable fragment (scFv) specific to a potent antileukemic substance, harringtonine (HT) (HT-scFv) that can be used in enzyme-linked immunosorbent assay (ELISA) for the quantitative analysis of HT in the plant genus Cephalotaxus. The variable heavy (VH) and light chain (VL) genes were directly cloned from the cDNA of the hybridoma cell line 1D2, which secretes monoclonal antibody against HT (MAb 1D2). These genes were then assembled with a flexible peptide linker, specifically (Gly4Ser)3, through splicing by overlap extension PCR. The resulting HT-scFv gene was expressed in Escherichia coli. The denatured HT-scFv, produced as inclusion bodies, was solubilized and refolded using a dilution method to restore its functionality as an antibody. Characterization of the HT-scFv demonstrated its high specificity for HT. Additionally, its remarkable properties facilitated the development of an ELISA for HT detection, achieving a limit of detection (LOD) of 12.2 ng/mL. Furthermore, validation analyses showed that the ELISA using HT-scFv exhibited good accuracy and reliability for the quantitative assessment of HT in C. harringtonia "Fastigiata." This study highlights the potential of HT-scFv as a valuable tool for ELISA in the quantitative analysis of HT.