冻干狂犬病疫苗热原检测在HL-60单核细胞激活实验中的方法转移和验证研究

The method transfer and validation of freeze-dried rabies vaccine pyrogen detection in HL-60 monocyte activation assay

目的研究人早幼粒白血病HL-60单核细胞激活实验在人狂犬疫苗冻干人用狂犬病疫苗热原检测中的可行性。方法将已建立的HL-60单核细胞激活实验进行实验室间方法转移,并进行方法验证;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13批冻干人用狂犬病疫苗中的回收率及其热原含量。结果以不同浓度内毒素标准品刺激HL-60细胞导致细胞分泌IL-6的量计算与内毒素浓度的线性,R^(2)均为0.95以上;计算最低检测限不超过0.125 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,考察方法的准确度;通过HL-60-IL-6检测13批冻干人用狂犬病疫苗,内毒素的回收率均在50%~200%;对4批热原检查、内毒素含量测定不合格的冻干人用狂犬病疫苗和附带的3批注射用水,该方法检测结果与家兔热原检查法和内毒素测定法的结果一致。结论以IL-6为检测指标的HL-60单核细胞激活实验适用于冻干人用狂犬病疫苗的热原物质检测。

Aim To study the application of HL-60 monocyte activation test in the pyrogen detection of freeze-dried rabies vaccine for human use.Methods The established HL-60 mononuclear cell activation test(MAT)was transferred between laboratories and the method was verified;referring to the interference test in the photometric method of the bacterial endotoxin test method of the Chinese Pharmacopoeia,HL-60-IL-6 MAT was used to detect the recovery and pyrogen content of 13 batches of freeze-dried rabies vaccine for human use.Results The linearity of the amount of IL-6 secreted by HL-60 cells,which stimulated by different concentrations of endotoxin standards was above 0.95;the calculated minimum detection limit was not more than 0.125 EU·mL^(-1);the recovery experiment with a solution containing 0.5 and 1.0 EU·mL^(-1) of endotoxin was performed to check the accuracy of the method.HL-60-IL-6 was used to detect 13 batches of Freeze-dried rabies vaccine for human use,and the recovery of endotoxin was between 50%to 200%.It was consistent that HL-60-IL-6 with pyrogens and endotoxin test for 4 batches of freeze-dried rabies vaccine for human use which pyrogens and endotoxin test failed and the 3 batches of water for injection.Conclusion The HL-60 MAT using IL-6 as a detection indicator is suitable for the detection of pyrogenic substances in freeze-dried rabies vaccine for human use.