Preparation and application of a specific single-chain variable fragment against artemether

Abstract

Artemether, an artemisinin derivative, is a component of the commonly used artemisinin-based combination therapy, artemether-lumefantrine. In this study, we cloned the VH and VL genes of a cell line (mAb 2G12E1) producing a monoclonal antibody specific to artemether, and used to construct a recombinant DNA of single-chain variable fragment (scFv). The scFv was constructed into prokaryotic expression vectors pET32a (+), pET22b (+), pGEX-2T, and pMAL-p5x, respectively. However, only the pMAL-p5x/scFv could be induced to express soluble scFv with comparable sensitivity and specificity to that of mAb 2G12E1. Based on the anti-artemether scFv, an indirect competitive enzyme-linked immunosorbent assay (icELISA) was developed. The 50% of inhibition concentration (IC₅₀) value and the working range based on IC₂₀ to IC₈₀ were 4.33 ng mL^-1 and 1.05-22.65 ng mL^-1, respectively. The artemether content in different drugs were determined by the developed icELISA, and the results were consistent to those determined by ultra performance liquid chromatography (UPLC). The anti-artemether scFv prepared in the current study could be a valuable genetically engineered antibody applied for artemether monitoring and specific binding mechanism studying.

Keywords: Artemether; Artemisinin-based combination therapies (ACTs); ELISA; Malaria; scFv.

Fig. 1

Structures of artemisinin and its analogs

Fig. 2

Deduced amino acid sequences of VH and VL regions of anti-artemether antibody

Fig. 3

Protein expression analysis (A), protein purification (B) and western blotting (C) further confirmed expression of anti-artemether scFv. (A) Lane 1, empty plasmid (pMAL-p5x) before induction; Lane 2, empty plasmid (pMAL-p5x) after induction; Lane 3, the supernatant of recombinant E. coli culture before induction; Lane 4, supernatant of recombinant E. coli culture after induction; Lane 5, total protein from recombinant E. coli before induction; Lane 6, total protein from recombinant E. coli after induction; M, Protein marker. (B) M, Protein marker; Lane 1-4, eluted protein samples. (C) Lane 1, empty plasmid (pMAL-p5x) after induction; Lane 2, total protein from recombinant E. coli after induction; Lane 3, eluted protein sample

Fig. 4

Binding activity of anti-artemether scFv to the antigen 9-hydroxyartemether-BSA

Fig. 5

Binding kinetics of the anti-artemether scFv measured by SPR

Fig. 6

Standard inhibition curve of artemether in icELISA format. B₀ and B are absorbance in the absence and presence of competitors, respectively. Concentration causing 50% inhibition by artemether was 4.33 ng mL⁻¹. Each value represents the mean of three replicates

Fig. 7

Bland-Altman bias plots for ELISA and UPLC. Quantitating artemether drugs concentration expressed as mg mL⁻¹. The solid line represents the bias between the assays, and the dashed lines represent the bias ± 1.96-s limits