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.
摘要：
蒿甲醚,青蒿素衍生物，是常用的青蒿素联合疗法的组成部分，蒿甲醚-本特林。在这项研究中,我们克隆了细胞系（mAb2G12E1）的VH和VL基因，产生了对蒿甲醚特异的单克隆抗体，用于构建单链可变片段(scFv)的重组DNA。将scFv构建到原核表达载体pET32a(+)中,pET22b(+),pGEX-2T,和pMAL-p5x，分别。然而,只有pMAL-p5x/scFv可以被诱导表达可溶性scFv，其敏感性和特异性与mAb2G12E1相当。基于抗蒿甲醚scFv，开发了一种间接竞争酶联免疫吸附测定（icELISA）。50%的抑制浓度(IC50)值和基于IC20至IC80的工作范围分别为4.33ngmL-1和1.05-22.65ngmL-1。通过开发的icELISA测定不同药物中的蒿甲醚含量，结果与超高效液相色谱法(UPLC)测定结果一致。本研究中制备的抗蒿甲醚scFv可能是一种有价值的基因工程抗体，可用于蒿甲醚的监测和特异性结合机制研究。
