Division of Molecular Medicine, Department of Research and Development, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok, Thailand.
Department of Biochemistry, Faculty of Sciences, Kasetsart University, Bangkok, Thailand.
Int J Mol Med. 2014 Mar;33(3):515-22. doi: 10.3892/ijmm.2014.1622. Epub 2014 Jan 13.
Macrophage migration inhibitory factor (MIF) is a pro-inflammatory cytokine, secreted from a variety of immune cells, that regulates innate and adaptive immune responses. Elevation of MIF levels in plasma correlates with the severity of inflammatory diseases in humans. Inhibition of MIF or its tautomerase activity ameliorates disease severity by reducing inflammatory responses. In this study, the human single-chain variable fragment (HuScFv) antibody specific to MIF was selected from the human antibody phage display library by using purified recombinant full-length human MIF (rMIF) as the target antigen. Monoclonal HuScFv was produced from phage-transformed bacteria and tested for their binding activities to rMIF by indirect enzyme-linked immunosorbent assay as well as to native MIF by western blot analysis and immunofluorescence assay. The HuScFv with highest binding signal to rMIF also inhibited the tautomerase activities of both rMIF and native MIF in human monoblastic leukemia (U937) cells in a dose-dependent manner. Mimotope searching and molecular docking concordantly demonstrated that the HuScFv interacted with Lys32 and Ile64 in the MIF tautomerase active site. To the best of our knowledge, this is the first study to focus on MIF-specific fully-human antibody fragment with a tautomerase-inhibitory effect that has potential to be developed as anti-inflammatory biomolecules for human use.
巨噬细胞移动抑制因子（MIF）是一种促炎细胞因子，由多种免疫细胞分泌，调节先天和适应性免疫反应。血浆中 MIF 水平的升高与人类炎症性疾病的严重程度相关。抑制 MIF 或其互变异构酶活性通过减少炎症反应来改善疾病严重程度。在这项研究中，用人重组全长人 MIF（rMIF）作为靶抗原，从人抗体噬菌体展示文库中筛选出针对 MIF 的人单链可变片段（HuScFv）抗体。从噬菌体转化的细菌中制备单克隆 HuScFv，并通过间接酶联免疫吸附试验以及 Western blot 分析和免疫荧光试验检测其与 rMIF 的结合活性和天然 MIF 的结合活性。与 rMIF 结合信号最强的 HuScFv 还能以剂量依赖的方式抑制人单核白血病（U937）细胞中 rMIF 和天然 MIF 的互变异构酶活性。模拟表位搜索和分子对接一致表明，HuScFv 与 MIF 互变异构酶活性部位的 Lys32 和 Ile64 相互作用。据我们所知，这是第一项研究针对具有互变异构酶抑制作用的 MIF 特异性全人抗体片段的研究，该抗体片段有可能被开发为用于人类的抗炎生物分子。
