Bai Xue, Zhang Lin, Hu Jin, Zhao Xiuxiu, Pan Jinheng, Deng Haiteng, Feng Shan
Key Laboratory of Structural Biology of Zhejiang Province, School of Life Sciences, Westlake University; Mass Spectrometry Core Facility, School of Life Sciences, Westlake University.
MOE Key Laboratory of Bioinformatics, Center for Synthetic and Systems Biology, School of Life Sciences, Tsinghua University.
J Vis Exp. 2019 Sep 17(151). doi: 10.3791/60063.
Elimination of harmful antibodies from organisms is a valuable approach for the intervention of antibody-associated diseases, such as Dengue hemorrhagic fever and autoimmune diseases. Since thousands of antibodies with different epitopes are circulating in blood, no universal method, except for the dual-functional conjugate of antigenic peptide and Fc-III mimetics (DCAF), was reported to target specific harmful antibodies. The development of DCAF molecules makes significant contribution to the progress of targeted therapy, which were demonstrated to eliminate the antibody dependent enhancement (ADE) effect in a Dengue virus (DENV) infection model and to boost the acetylcholine receptor activity in a myasthenia gravis model. Here, we describe a protocol for the synthesis of a DCAF molecule (DCAF1), which can selectively block 4G2 antibody to attenuate ADE effect during Dengue virus infection, and illustrate the binding of DCAF1 to 4G2 antibody by an ELISA assay. In our method, DCAF1 is synthesized by the conjugation of a hydrazine derivative of a Fc-III peptide and a recombinant expressed long α-helix with antigenic sequence through native chemical ligation (NCL). This protocol has been successfully applied to DCAF1 as well as other DCAF molecules for targeting their cognate antibodies.
从生物体中清除有害抗体是干预抗体相关疾病（如登革出血热和自身免疫性疾病）的一种有价值的方法。由于数千种具有不同表位的抗体在血液中循环，除了抗原肽与Fc-III模拟物的双功能缀合物（DCAF）外，尚无针对特定有害抗体的通用方法被报道。DCAF分子的开发为靶向治疗的进展做出了重大贡献，在登革病毒（DENV）感染模型中，DCAF分子被证明可消除抗体依赖性增强（ADE）效应，在重症肌无力模型中可增强乙酰胆碱受体活性。在此，我们描述了一种DCAF分子（DCAF1）的合成方案，该分子可选择性阻断4G2抗体，以减轻登革病毒感染期间的ADE效应，并通过ELISA测定法阐明DCAF1与4G2抗体的结合。在我们的方法中，DCAF1是通过Fc-III肽的肼衍生物与具有抗原序列的重组表达长α-螺旋通过天然化学连接（NCL）进行缀合而合成的。该方案已成功应用于DCAF1以及其他靶向其同源抗体的DCAF分子。
