Duensing Thomas D, Watson Susan R
Cold Spring Harb Protoc. 2018 Jan 2;2018(1):2018/1/pdb.prot093807. doi: 10.1101/pdb.prot093807.
A useful feature of therapeutic antibodies is the ability to kill the cells to which they bind. Antibodies are capable of mediating cell killing in a variety of ways. Apoptosis, complement-mediated mechanisms, and antibody-dependent cellular cytotoxicity are all effects that can be assayed to characterize lead antibody candidates. Extensive, multidose characterizations of a series of candidates can be performed in a short amount of time using assays developed for high-throughput flow cytometry systems. Here, we describe a simple multiplexed flow assay performed using Annexin V and propidium iodide that measures an early marker of apoptosis. When cells enter apoptosis, phosphatidyl serine (PS), which is normally found on the inside of the cytoplasmic membrane, is found on the extracellular surface of the membrane, thus revealing Annexin V-binding sites. Because binding of Annexin V to PS is calcium dependent, the buffers used for this assay must contain 1 mm calcium. The calcium dependence can also be used to test whether the Annexin V staining is specific. Thus, if the staining is performed in the presence of 1 mm EDTA, binding of Annexin V should be inhibited. The addition of propidium iodide allows subsequent stages of apoptosis and eventual cell death to be distinguished. For flow cytometry, this assay is best performed on suspension cells.
治疗性抗体的一个有用特性是能够杀死它们所结合的细胞。抗体能够通过多种方式介导细胞杀伤。凋亡、补体介导的机制以及抗体依赖性细胞毒性都是可以检测的效应，用于表征潜在的先导抗体。使用为高通量流式细胞术系统开发的检测方法，可以在短时间内对一系列候选抗体进行广泛的多剂量表征。在此，我们描述一种使用膜联蛋白V和碘化丙啶进行的简单多重流式检测，该检测可测量凋亡的早期标志物。当细胞进入凋亡时，通常位于细胞质膜内侧的磷脂酰丝氨酸（PS）会出现在膜的细胞外表面，从而暴露出膜联蛋白V的结合位点。由于膜联蛋白V与PS的结合是钙依赖性的，因此该检测所用的缓冲液必须含有1 mM的钙。钙依赖性还可用于测试膜联蛋白V染色是否具有特异性。因此，如果在1 mM乙二胺四乙酸（EDTA）存在的情况下进行染色，膜联蛋白V的结合应受到抑制。加入碘化丙啶可以区分凋亡的后续阶段和最终的细胞死亡。对于流式细胞术，该检测最好在悬浮细胞上进行。
