中文摘要：

目的建立一个可靠的、可供临床应用检测的rAAV2一KAL残余HEK293细胞宿主蛋白（host cell protein，HCP）的测试方法。方法Western blot方法鉴定rAAV，ELISA方法确定最佳线性范围及最低检测限，验证准确度及精密度。本实验连续生产并用二次氯化铯纯化3批rAAV2一KAL，使用ELISA方法检测rAAV2-KAL所含有的HCP含量，验证超速离心纯化工艺中的适用性。结果建立的ELSA方法的最佳线性范围为4—200ng·mL-1，最低检测为4ng·mL-1；不同浓度的HEK293细胞蛋白抗原回收率77．0％～115．0％，准确度较好；变异系数为8．2％～15．4％均低于20％，显示准确度和精密度均良好；制备的3批rAAV2-KAL经过二次CsCl纯化后，HCP含量均低于100ng·mL-1，显示该纯化工艺可有效去除HCP。结论本实验成功建立HEK293细胞残余蛋白含量检测的双抗体夹心法ELSA方法，可用于rAAV病毒载体中HEK293宿主细胞蛋白残余含量的检测。

英文摘要：

OBJECTIVE To develop a reliable method to measure host cell protein （HCP）content of HEK293 cells in rAAV2- KAL vectors for clinic application. METHODS The rAAV2-KAL vectors used for this study was purified by CsC1 ultracentrifugation twice, then were subjected to by Western blot. The optimal linear range, minimum detection limit, and the accuracy and precision were verified by ELISA. Three batches of rAAV2-KAL were prepared by CsC1 method. The change of HCP content during purification was determined to verify the method suitability and reliabilities. RESULTS The optimal linear range of the method developed was 4 - 200 ng· mL-1 ,while the minimum detection limit of 4 ng· mL-1. The recovery rates of HEK293 cell protein at various concentrations were at range of 77.0% - 115.0% ,with a coefficient of variation of less than 20%. The HCP contents in three batches of rAAV2-KAL were less than 100 ng·mL-l, all data taken together indicated that HCP was effectively removed by CsCl ultracentrifugation. CON- CLUSION A reliable ELISA assay for residual host cell protein of HEK293 cells is successfully developed, which might be used for determination of HCP content in CsC1 uhracentrifugation purified rAAV2-KAL for clinical application.