微量动态浊度法检测细菌内毒素方法的建立与验证

Establishment and Verification of the Method for Detecting Bacterial Endotoxins by the Micro-kinetic Turbidimetric Method

关键词:  细菌内毒素检查法  动态浊度法  鲎试剂  替代方法

Key words:  bacterial endotoxin test method  dynamic turbidity method  limulus reagent  alternative method

摘要:

目的 建立使用微量动态浊度法检测细菌内毒素的方法，并进行验证。方法 采用动态浊度法鲎试剂，每孔样品和鲎试剂加样量25 μL，检测波长405 nm，预设OD值0.03，使用半孔酶标板检测，并经过4个实验室协作验证方法的准确度、重现性、线性和范围、耐用性以及重组新型冠状病毒疫苗、重组新型冠状病毒疫苗(5型腺病毒载体)、重组乙型肝炎疫苗、麻腮风联合减毒疫苗、双价肾综合征出血热灭活疫苗、四价流感病毒裂解疫苗共计6种疫苗的品种适用性。结果 高、中、低3个剂量组在4个实验室的共计24次试验总平均回收率分别为100.8%，150.0%，125.3%，RSD值分别为5.3%，11.0%，20.7%；标准曲线在0.01~10 EU·mL^-1内相关系数平均值为0.990，耐用度考察2个来源的鲎试剂对检测的影响符合规定；6个品种的共计18批样品的干扰试验回收率均在50%~200%，干扰试验符合规定。结论 建立了使用微量动态浊度法检测细菌内毒素的方法，该方法具有良好的准确度、重现性、线性和范围、耐用性，并且在6种生物制品中具有良好的品种适用性。该方法可用于常规样品的细菌内毒素检测。

Abstract:

OBJECTIVE To establish and verify a detection method for bacterial endotoxins using the micro-kinetic turbidimetric method. METHODS The dynamic turbidimetric limulus reagent was used, the sample volume of each well and the limulus reagent 25 μL was used, the detection wavelength was 405 nm, the onset OD value was 0.03, and the half-well microtiter plate was used. The accuracy, reproducibility, linearity, range, and durability of the method had been verified by 4 laboratories collaboratively. A total of 6 vaccines included recombinant novel coronavirus vaccine, recombinant novel coronavirus vaccine(type 5 adenovirus vector), recombinant hepatitis B vaccine, combined attenuated vaccine for measles and mumps, inactivated bivalent hemorrhagic fever with renal syndrome vaccine, and influenza vaccines were being studied for method applicability. RESULTS The total average recovery rates of 24 trials in 4 laboratories in the high, medium and low dose groups were 100.8%, 150.0% and 125.3%, respectively, the RSD values were 5.3%, 11.0%, and 20.7%. The standard curve had an average correlation coefficient of 0.990 in the range of 0.01-10 EU·mL^-1. The durability study was investigated. The influence of the two sources of limulus reagent on the detection was in compliance with the regulations. The recovery rates of the interference test for a total of 18 batches of samples of 6 varieties were all within between 50% and 200%, the interference test met the requirements. CONCLUSION A method for detecting bacterial endotoxins using micro-kinetic turbidimetry has been established. This method has good accuracy, reproducibility, linearity and range, durability, and has good variety applicability among 6 biological products. This method can be used for bacterial endotoxin detection of conventional samples.