乙型脑炎减毒活疫苗细菌内毒素凝胶法的检测

Gel-Clot Method of Bacterial Endotoxin Test for Japanese Encephalitis Live Attenuated Vaccine

目的建立乙型脑炎减毒活疫苗细菌内毒素检测方法和质量标准,检测样品中的内毒素含量。方法中国药典2010年版三部细菌内毒素检测法(凝胶限度试验、凝胶半定量试验)。结果选用λ为0.5 EU/ml的鲎试剂,采用凝胶限度法对乙型脑炎减毒活疫苗进行细菌内毒素检查,样品经稀释至80倍或以上稀释度后对内毒素检查均无干扰,确定该疫苗的内毒素限度值为40 EU/ml[即20 EU/剂(0.5 ml)]。凝胶限度试验结果显示,疫苗每1 ml中含内毒素的量均小于40EU,即每1次人用剂量(0.5 ml)中含内毒素的量均小于20 EU。凝胶半定量测定结果显示,1人份规格疫苗中含内毒素的量为2.50～10.00 EU/ml(平均5.36 EU/ml);5人份规格疫苗中含内毒素的量为1.50～6.00 EU/ml(平均3.02 EU/ml)。结论凝胶法两种试验均可用于乙型脑炎减毒活疫苗细菌内毒素检测,与凝胶半定量试验相比,凝胶限度试验更为简便、且易于操作;采用凝胶限度法本研究建立了乙型脑炎减毒活疫苗细菌内毒素检测方法和质量标准;凝胶半定量法测定结果用准确数据显示该疫苗的内毒素含量均在人体安全限度以内。

Objective To establish the inspection methods and quality criteria for bacterial endotoxin of Japanese encephalitis（JE） live attenuated vaccine and detect the endotoxin contents of the test vaccines.Method Tests were conducted according to the bacterial endotoxin test（BET） methods（gel-clot limit test and gel-clot assay） from Pharmacopoeia of People＇s Republic of China（2010） Volume Ⅲ.Results The bacterial endotoxin of JE live attenuated vaccine was detected by gel-clot limit test by using amebocyte lysate with 0.5 EU/ml sensitivity.No interference of samples to the endotoxin test was observed when the samples were diluted to 80-fold or above dilution,and therefore 40 EU/ml（20 EU in each 0.5ml human dose） was confirmed as the detection limit for endotoxin of JE live attenuated vaccine.The results of gel-clot limit test revealed that the endotoxin contents of test vaccines were all less than 40 EU in 1 ml,namely less than 20 EU in each 0.5 ml human dose.The results of gel-clot assay showed that the endotoxin contents of test vaccines were 2.50-10.00 EU/ml（mean=5.36 EU/ml） for JE live attenuated vaccine containing 1 dose and 1.50-6.00 EU/ml（mean=3.02 EU/ml） for JE live attenuated vaccine containing 5 doses.Conclusions Both gel-clot limit test and gel-clot assay can be used for the bacterial endotoxin test of JE live attenuated vaccine.Gel-clot limit test is more convenient and liable to operation compared with gel-clot assay.The inspection methods and quality criteria for bacterial endotoxin test of JE vaccine（live） have been established in this study by using gel-clot limit test method.The results of gel-clot assay quantitatively indicate that the endotoxin contents of JE live attenuated vaccine are all within the safety range for human body.