基于THP1-Blue^(TM) NF-κB报告基因法的体外热原检测方法开发与验证  

Development and validation of pyrogen determination in vitro based on THP1-Blue^(TM)NF-κB reporter gene assay

作　　者：吴彦霖[1] 张铭露 杨泽岸 陈宏宇 张媛[1] WU Yan-lin;ZHANG Ming-lu;YANG Ze-an;CHEN Hong-yu;ZHANG Yuan(China Institute of Food and Drug Control,Beijing 100050,China;Henan Institute of Drug and Medical Devices,Zhengzhou 450018,China;Beijing Institute of Petrochemical Technology,Beijing 102617,China)

机构地区：[1]中国食品药品检定研究院,北京100050 [2]河南省药品医疗器械检验院,郑州450018 [3]北京石油化工学院,北京102617

出　　处：《中国新药杂志》2024年第22期2331-2337,共7页Chinese Journal of New Drugs

基　　金：国家“重大新药创制”科技重大专项资助项目:创新生物技术药评价及标准化关键技术研究(2018ZX09101001)。

摘　　要：目的:利用导入核因子κB(nuclear factor kappa-B,NF-κB)报告基因和分泌型胚胎碱性磷酸酶(secreted alkaline phosphatase,SEAP)活性的人单核细胞白血病细胞系THP-1细胞,建立一种可用于体外热原检测的方法,并考察方法的可行性和品种适用性。方法:参照《中华人民共和国药典》四部通则9101分析方法验证和9301注射剂安全检查法应用指导原则(附单核细胞活化反应测定法),以细菌内毒素国家标准品作为基准,对THP1-BlueTM NF-κB报告基因法进行专属性、精密度、耐用性等方法学研究;以精密度和阈值作为方法可行性分析的判断指标;并进行品种适用性研究。结果:所建立的方法符合《中华人民共和国药典》9301中单核细胞活化反应测定法的要求,R2均为0.999以上;计算最低检测限0.03 EU·mL^(-1);以内毒素含量为0.5,1.0 EU·mL^(-1)的溶液做回收实验,方法的准确度分别为101%和106%;通过本方法完成人凝血酶原复合物、冻干人纤维蛋白原、b型流感、23价肺炎、牛痘疫苗的品种适用性研究。结论:THP1-Blue^(TM) NF-κB报告基因法满足单核细胞激活实验要求,可用于体外热原物质检测。Objective:To establish a method for detection of pyrogens using THP-1 cells with NF-κB reporter gene and SEAP activity,and investigate the feasibility and species applicability of the method in vitro.Methods:With reference to the guidelines of the general rules of ChP,9101 Guidelines for validation of analytical methods and 9301 Guidelines for application of injection safety checking method(with monocyte activation reaction assay),and using the national standard product of bacterial endotoxin as the benchmark,a methodological study was conducted on the THP1-Blue^(TM)NF-κB reporter gene assay to examine its methodic feasibility in terms of appropriateness,precision,and durability.Precision and threshold were used as the judgment indicators for the method feasibility analysis.A study was also conducted on the applicability of the assay to different varieties.Results:According to 9301 Guidelines for monocyte activation reaction assay in ChP,the established method met the requirements of monocyte activation reaction assay,and the linear R~2 was above 0.999.The lowest detection limit was calculated as 0.03 EU·mL^(-1),and the accuracy of the method was 101%and 106%for the recovery experiments using the solutions with endotoxin content of 0.5 and 1.0 EU·mL^(-1),respectively.The method was successfully used to study the suitability of human prothrombin complex,lyophilized human fibrinogen,influenza b,23-valent pneumonia and cowpox vaccines.Conclusion:The THP1-Blue^(TM)NF-κB reporter gene method meets the requirements of monocyte activation assay and can be used for detection of pyrogenic substances in vitro.

关 键 词：单核细胞激活实验 热原检测 THP1-Blue^(TM)NF-κB 报告基因法 

分 类 号：R917[医药卫生—药物分析学]