Comparison of single-chain variable fragments and monoclonal antibody against dihydroartemisinin

Abstract

Immunoassay relies on antibodies, but traditional antibodies such as monoclonal antibody (mAb) require animal immunization and complex procedures. Single-chain variable fragment (scFv) becomes a potential alternative to mAb with advantages of the low cost, rapid and easy prepared. In the present study, we prepared scFvs against dihydroartemisinin (DHA) based on E. coli and HEK293T cell expression system, named MBP-scFv and scFv-Fc, respectively. Their properties were compared with the parent mAb. The calculated affinity constants of mAb, MBP-scFv and scFv-Fc were 2.1 × 10⁸ L mol⁻¹, 2.2 × 10⁷ L mol⁻¹ and 1.6 × 10⁸ L mol⁻¹, respectively. The half inhibitory concentration (IC₅₀) of mAb, MBP-scFv and scFv-Fc were 1.16 ng mL⁻¹, 2.15 ng mL⁻¹ and 6.57 ng mL⁻¹, respectively. Both the scFv showed less sensitive than the mAb based on the IC₅₀. The cross-reactivities of MBP-scFv for artemisinin and artesunate exhibited similarities to the mAb, yet the cross-reactivities of scFv-Fc for these compounds exceeded those of the mAb significantly. The stability of the scFvs was ascertained to be maintained for over 5 days at room temperature, and for more than a month at both 4 °C and − 20 °C. After that, the indirect competitive enzyme-linked immunosorbent assays (icELISAs) based on the scFv from E. coli were used to detect the DHA content in eight drug samples, and the result was consistent with ultra-performance liquid chromatography simultaneously. Although scFv can be used for quantitative determination of drugs, but it still cannot completely replace mAb in immunoassay without evolution and modification.