单核细胞激活实验在疫苗类生物制品热原检测中的应用研究

Application of Monocyte Activation Test in the Pyrogen Detection of Vaccine

目的:研究人早幼粒白血病HL-60单核细胞激活实验在疫苗类生物制品热原检测中的应用。方法:将HL-60单核细胞分别与不同浓度热原(内毒素、酵母多糖和脂壁酸)标准品溶液、样品溶液进行孵育,酶联免疫吸附试验(ELISA)检测孵育体系中IL-6的含量。以热原标准品浓度为横坐标,IL-6量为纵坐标做标准曲线,将样品测得的IL-6值代入该标准曲线,计算样品中热原的含量。选取处于标准曲线中间点的热原浓度,作为样品干扰试验中添加的热原浓度,参照《中国药典》细菌内毒素检查法中光度测定法中的干扰试验,用HL-60/IL-6单核细胞激活实验检测三种热原在流感疫苗、冻干甲型肝炎减毒活疫苗、冻干人用狂犬病疫苗、麻腮风联合减毒活疫苗、麻疹腮腺炎联合减毒活疫苗、双价肾综合征出血热灭活疫苗和A群C群脑膜炎球菌多糖疫苗7种疫苗中的回收率及其热原含量。结果:1不同浓度内毒素、酵母多糖和脂壁酸三种热原标准品刺激HL-60细胞后,该细胞分泌IL-6的量与热原浓度线性良好,R^2分别为0.996,0.993,0.997。2该方法测定三种热原在以上7种疫苗中的回收率,回收率均在50%~200%之间。3以上方法测2个厂家10个批号A群C群脑膜炎疫苗中的内毒素含量,厂家A的6批样品内毒素平均值为134.73 EU·ml^(-1),厂家B的4批样品内毒素平均值为40.75 EU·ml^(-1)。该方法检测其他6种疫苗中热原含量,结果均为阴性。结论:该方法适用于以上7种疫苗样品的热原检测。

Objective：To study the application of HL-60 monocyte activation test in the pyrogen detection of vaccines.Methods：HL-60 cells were incubated with the standard pyrogen（endotoxin,zymosan and lipoteichoic acid） solution and the sample solution at different concentrations,and then IL-6 level was detected using ELISA kit.According to the concentration of standard pyrogen solution and the level of IL-6,the standard curve and linear relationship were obtained.Based on the IL-6 value from the sample solution,the pyrogen content of the sample was calculated.The standard pyrogen solution at middle concentration of the standard curve was used as the interference sample,and the interference assay was performed referring to the bacterial endotoxin test in Chinese pharmacopoeia.The pyrogen recovery rate and the content were detected using HL-60/IL-6 assay in seven kinds of vaccines such as influenza vaccine（Split Virion）,inactivated;hepatitis A（live） vaccine,freeze-dried;rabies vaccine（Vero Cell） for human use,freeze-dried;lyophilized human measles and rubella combined vaccine,live;measles and mumps combined vaccine,live;haemorrhagic fever with renal syndrome bivalent vaccine,inactived;group A and C meningococcal polysaccharide vaccine.Results：1 The linear relationship between the pyrogen concentration and the IL-6 level was good,and R2 value was 0.996,0.993 and 0.997,respectively.2 The recovery rate of pyrogen in the above vaccines ranged from 50% to 200%.3 The endotoxin concentration of 10 batches of group A and C meningococcal polysaccharide vaccine was detected using HL-60/IL-6 assay,and the average value of 6 batches from factory A was 134.73 EU·ml^-1,and that of 4 batches from factory B was 40.75 EU·ml^-1.The pyrogen in the other vaccines was all negative.Conclusion：The HL-60/IL-6 assay is suitable for the pyrogen detection of the seven kinds of vaccines.