School of Pharmacy and Food Engineering, Wuyi University, Jiangmen, Guangdong 529020, China.
School of Pharmacy and Food Engineering, Wuyi University, Jiangmen, Guangdong 529020, China; Key Laboratory of High-Quality Formation and Utilization of Dao-Di Herbs, Guangxi Botanical Garden of Medicinal Plants, Nanning 530023, China.
J Immunol Methods. 2024 Sep;532:113728. doi: 10.1016/j.jim.2024.113728. Epub 2024 Jul 24.
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 × 10 L mol, 2.2 × 10 L mol and 1.6 × 10 L mol, respectively. The half inhibitory concentration (IC) of mAb, MBP-scFv and scFv-Fc were 1.16 ng mL, 2.15 ng mL and 6.57 ng mL, respectively. Both the scFv showed less sensitive than the mAb based on the IC. 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.
免疫分析依赖于抗体，但传统抗体，如单克隆抗体（mAb），需要动物免疫和复杂的程序。单链可变片段（scFv）成为 mAb 的潜在替代品，具有成本低、快速、易于制备的优点。在本研究中，我们基于大肠杆菌和 HEK293T 细胞表达系统，制备了针对二氢青蒿素（DHA）的 scFv，分别命名为 MBP-scFv 和 scFv-Fc。比较了它们与亲本 mAb 的性质。mAb、MBP-scFv 和 scFv-Fc 的计算亲和力常数分别为 2.1×10^L/mol、2.2×10^L/mol 和 1.6×10^L/mol。mAb、MBP-scFv 和 scFv-Fc 的半抑制浓度（IC）分别为 1.16ng/mL、2.15ng/mL 和 6.57ng/mL。基于 IC，两种 scFv 的灵敏度均低于 mAb。MBP-scFv 对青蒿素和青蒿琥酯的交叉反应性与 mAb 相似，但 scFv-Fc 对这些化合物的交叉反应性明显高于 mAb。确定 scFv 的稳定性在室温下可保持超过 5 天，在 4°C 和-20°C 下可保持超过一个月。之后，基于大肠杆菌 scFv 的间接竞争酶联免疫吸附试验（icELISA）用于检测 8 种药物样品中的 DHA 含量，结果与超高效液相色谱法一致。虽然 scFv 可用于药物的定量测定，但在没有进化和修饰的情况下，它仍然不能完全替代免疫分析中的 mAb。
