目的 建立肠道病毒71型（enterovirus 71, EV71）纯度工作参考品，应用于高效液相色谱法检验EV71灭活疫苗（Vero细胞）原液纯度过程中的质量控制，并研究该工作参考品的长期稳定性。方法 将EV71接种Vero细胞，经纯化、灭活的病毒原液分装后作为EV71工作参考品，并检定主要评价指标，包括无菌性及蛋白质含量等。采用高效液相色谱法对EV71工作参考品的纯度进行标定，并将参考品于2～8 ℃条件下放置72个月，分别在不同时间进行纯度检测，考察其长期稳定性。结果 建立的EV71工作参考品的无菌性及蛋白质含量等检查结果均符合规定，其标定纯度为98.02%～100.00%，满足工作参考品的质量控制要求。在2～8 ℃条件下放置72个月内病毒蛋白峰峰形保持一致，保留时间为6.861～7.473 min，相对标准偏差为2.6%；纯度的相对标准偏差为0.1%，纯度未见明显改变。结论 建立的EV71纯度工作参考品长期稳定性良好，可用于EV71灭活疫苗（Vero细胞）原液纯度的检测。

Objective To establish an enterovirus 71 (EV71) working reference for quality control in the purity test of inactivated EV71 vaccine (Vero cell) bulk by high-performance liquid chromatography (HPLC), and to study the long-term stability of this working reference.Methods The EV71 working reference was prepared from the inactivated and purified virus bulk of EV71 inoculated in Vero cells, and was subjected to quality control tests, including sterility and protein content determination. The purity of EV71 working reference was calibrated using HPLC. To evaluate its long-term stability, the reference was stored at 2-8 °C for 72 months, with purity assays conducted at specified time points.Results The sterility and protein content of EV71 working reference met the specified requirements. Its calibrated purity was determined to be 98.02%-100.00%, meeting the quality control criteria for working reference. After storage at 2-8 °C for 72 months, the viral protein peak profile remained consistent, with retention time of 6.861-7.473 min and relative standard deviation (RSD) of 2.6%. The purity RSD was 0.1%, with no significant changes observed.Conclusion The established working reference for EV71 purity exhibits good long-term stability and can be used for the purity testing of inactivated EV71 vaccine (Vero cell) bulk.