Broyer Lucile, Goetsch Liliane, Broussas Matthieu
Experimental Oncology Department, Centre d'Immunologie Pierre-Fabre, Saint Julien-en-Genevois, France.
Methods Mol Biol. 2013;988:319-29. doi: 10.1007/978-1-62703-327-5_20.
One of the major issues for antibody treatment is enhancement of efficacy. Recent studies have highlighted the important role of effector functions in improvement of antibody therapy. Among effector functions, complement-dependent cytotoxicity (CDC), which induces cell lysis by a cascade of activation triggered by the binding of C1q subunits to the Fc regions of antibodies bound to the cell surface, is part of the mechanism of several antibody therapies. CDC can be modulated by either Fc isotype engineering or Fc genetic mutations or Fc glycosylation profile modifications. To evaluate the impact of such modifications on CDC, we describe a luminescence method based on ATP measurement to estimate tumor damaged cells and a flow cytometry method to evaluate the binding of C1q on the Fc region and the binding of C4b on cell surface. The luminescence method coupled with complement protein analysis by flow cytometry encompasses all needed methods to evaluate antibody ability to trigger CDC.
抗体治疗的主要问题之一是提高疗效。最近的研究强调了效应功能在改善抗体治疗中的重要作用。在效应功能中，补体依赖性细胞毒性（CDC）是几种抗体治疗机制的一部分，它通过C1q亚基与结合在细胞表面的抗体的Fc区域结合触发的一系列激活来诱导细胞裂解。CDC可以通过Fc同种型工程、Fc基因突变或Fc糖基化谱修饰来调节。为了评估这些修饰对CDC的影响，我们描述了一种基于ATP测量来估计肿瘤受损细胞的发光方法和一种用于评估C1q在Fc区域的结合以及C4b在细胞表面的结合的流式细胞术方法。结合流式细胞术进行补体蛋白分析的发光方法涵盖了评估抗体触发CDC能力所需的所有方法。
