正在评估一种Coxsackievirus A21 (CVA21) 的原型菌株，作为一个溶瘤病毒免疫疗法。CVA21 优先裂解那些表达细胞间黏附分子 1 的细胞，其中包括某些类型的肿瘤细胞。CVA21 具有由 60 个蛋白亚单位组成的二十面体外壳结构，包裹着一个具有 30nm 粒径尺寸的病毒 RNA 基因组。快速且强大的分析方法，能够量化 CVA21 的总病毒、空病毒和满病毒粒子，是支持过程开发、满足监管要求和验证制造过程的重要保证。在本研究中，我们利用自行生产的抗体，演示了所有四种 CVA21 外壳蛋白 VP1、VP2、VP3 和 VP4，以及VP0，这是空粒子的替代物的检测方法。利用这些抗体，开发了一种自动化和定量的毛细管电泳西方印迹（Simple Western）试验，通过 VP1 量化 CVA21 总颗粒，通过 VP0 量化空颗粒、通过 VP0 和 VP4 量化空颗粒到满颗粒的相对比例、以及通过 VP0 和 VP1 量化空颗粒到总颗粒的绝对比例。最后，这种 Simple Western 方法被用作支持 CVA21 细胞培养和纯化过程的优化，作为一个高通量的分析工具，以快速制定过程决策。

A prototype strain of Coxsackievirus A21 (CVA21) is being evaluated as an oncolytic virus immunotherapy. CVA21 preferentially lyses cells that upregulate the expression of intercellular adhesion molecule 1, which includes some types of tumor cells. CVA21 has an icosahedral capsid structure made up of 60 protein subunits encapsidating a viral RNA genome with a particle diameter size of 30 nm. Rapid and robust analytical methods to quantify CVA21 total, empty, and full virus particles are important to support the process development, meet regulatory requirements, and validate manufacturing processes. In this study, we demonstrate the detection of all four CVA21 capsid proteins, VP1, VP2, VP3, and VP4, as well as VP0, a surrogate for empty particles, using in-house-generated antibodies. An automated and quantitative capillary Western blot assay, Simple Western, was developed using these antibodies to quantify CVA21 total particles through VP1, empty particles through VP0, relative ratio of empty to full particles through VP0 and VP4, and the absolute ratio of empty to total particles through VP0 and VP1. Finally, this Simple Western method was used to support CVA21 cell culture and purification process optimization as a high-throughput analytical tool to make rapid process decisions.