吸附无细胞百(三组分)白破联合疫苗内毒素定量检测方法的建立及验证

Establishment and verification of quantitative detection method for endotoxin of diphtheria,tetanus and acellular pertussis(three components)combined vaccine,adsorbed

目的建立吸附无细胞百日咳(三组分)-白喉-破伤风联合疫苗﹝diphtheria, tetanus and acellular pertussis (three components) combined vaccine, adsorbed, DTacP﹞内毒素含量的定量检测方法, 并对其进行验证。方法采用动态显色法(美洲鲎试剂)检测DTacP的内毒素含量, 并对该方法的线性与范围、专属性、准确度、中间精密度、重复性、定量限进行验证, 并与凝胶法结果进行比较。结果动态显色法的线性相关系数的绝对值大于0.99;DTacP稀释100倍和1 000倍, 对试验均无干扰作用;3批DTacP内毒素检测结果的准确度回收率分别为56%、75%、80%, 重复性的变异系数分别为5.59%、7.14%、5.81%;不同操作人员在不同时间检测3批DTacP中内毒素结果的变异系数分别为15.93%、8.48%和7.43%;将光密度反应阈值30毫吸光度单位增减3毫吸光度单位, 耐用性的相对偏差均小于15%。DTacP中内毒素含量检测的定量限为30内毒素单位(endotoxin unit, EU)/ml。动态显色法检测3批DTacP中内毒素含量的检测结果与凝胶法结果一致, 均小于限定值200 EU/ml。结论建立了定量检测DTacP中内毒素含量的动态显色法, 该法具有良好的线性、专属性、准确度、中间精密度以及重复性, 可用于DTacP中内毒素含量的定量检测。

ObjectiveTo establish and verify a quantitative detection method for endotoxin content of diphtheria,tetanus and acellular pertussis(three components)combined vaccine,adsorbed(DTacP).MethodsA kinetic chromogenic assay(Limulus amebocyte lysate)was established to detect the endotoxin content of DTacP,and the linearity and range,specificity,accuracy,intermediate precision,repeatability,and limit of quantitation of the method were verified,and the results were compared with those of gel method.ResultsThe absolute value of the linear correlation coefficient of the method was more than 0.99.DTacP diluted 100 and 1000 times had no interference on the experiment.The accuracy recovery rates of 3 batches of DTacP were 56%,75%and 80%,respectively.The coefficients of variation(CV)of repeatability were 5.59%,7.14%and 5.81%,respectively.The intermediate precision of DTacP was verified by different operators at different times and the CV values of three batches of DTacP were 15.93%,8.48%and 7.43%,respectively.Adjusting the optical density threshold of 30 mili-absorbance unit(mAbs)by±3 mAbs,the relative deviation of durability was less than 15%.The limit of quantitation of DTacP was 30 endotoxin unit(EU)/ml.The bacterial endotoxin content of 3 batches of DTacP by kinetic chromogenic assay was consistent with that of gel method,all less than the bacterial endotoxin limit of 200 EU/ml.ConclusionA quantitative detection method for endotoxin content of DTacP by kinetic chromogenic assay is established,which has good linearity,specificity,accuracy,intermediate precision and repeatability,and can be used for quantitative detection of endotoxin content of DTacP.