摘要
目的:评价有机溶剂/表面活性剂(solvent and detergent,S/D)法灭活后S/D残留量对严重急性呼吸综合征冠状病毒2(SARS-CoV-2)的假病毒中和抗体检测方法(pseudotyped virus based neutralization assay,PBNA)的影响。方法:在严重急性呼吸综合征(severe acute respiratory syndrome,SARS)和2019冠状病毒病(coronavirus disease,COVID-19)患者恢复期血浆中加入0.1~10倍《中华人民共和国药典》(简称《中国药典》)规定残留标准[100μg·mL^-1聚山梨酯80(吐温80)+10μg·mL^-1磷酸三丁酯(TNBP)]浓度的S/D,模拟灭活后S/D残留量。用PBNA检测加入S/D前后的中和抗体水平,比较S/D对中和方法的影响。将不同浓度的S/D加入到SARS-CoV和SARS-CoV-2假病毒中进行灭活处理1、2和6 h,检测假病毒的滴度,评价S/D对冠状病毒假病毒的灭活效果。结果:当S/D残留低于《中国药典》残留上限(100μg·mL^-1吐温80+10μg·mL^-1 TNBP)时,SARS-CoV-2中和抗体EC50平均值与无S/D组的PBNA检测结果无统计学差异;当S/D残留大于2.5倍《中国药典》残留标准时,S/D对PBNA有显著影响。含有10 mg·mL^-1吐温80+1 mg·mL^-1 TNBP的S/D试剂,室温静置1 h即可以灭活SARSCoV和SARS-CoV-2假病毒,假病毒滴度下降分别达到5.11和4.80 log滴度,其灭活效果随着S/D的浓度下降而下降,S/D浓度为1%时无灭活效果。结论:S/D残留量对SARS-CoV-2的PBNA有显著影响,为保障PBNA的检测准确性,血浆中S/D残留量应不超过《中国药典》规定的标准,S/D试剂具有灭活2种冠状病毒假病毒的作用。
Objective:To evaluate the influence of residual solvent and detergent(S/D),which is used to inactivate SARS-CoV-2 in convalescent plasma(CP),on pseudotyped virus based neutralization assay(PBNA).Methods:To simulate S/D residue after inactivation,S/D from 0.1 to 10-fold amounts of what is listed in Pharmacopoeia of the People’s Republic of China(Chinese Pharmacopoeia)[100μg·mL^-1 polysorbate 80(Tween 80)+10μg·mL^-1 tributyl phosphate(TNBP)]were added into CPs from SARS(severe acute respiratory syndrome,SARS)or COVID-19(coronavirus disease,COVID-19)infected individuals.The titers of neutralizing antibody before and after S/D addition were detected by PBNA.The influence of S/D on the detection was investigated.After 1,2 and 6 hours of treatment,the titers of pseudotyped virus were detected to evaluate the inactivation effect of S/D on coronavirus pseudotyped viruses.Results:When S/D residue did not exceed the residue standard(100μg·mL^-1 Tween 80+10μg·mL^-1 TNBP)stipulated in Chinese Pharmacopoeia,no significant difference was observed between the S/D and control groups for EC50 values of SARS-CoV-2 neutralizing antibody.When S/D residue exceeded the 2.5 folds of residue standard,a significant inhibitory effect was presented on neutralizing antibody detection.S/D containing 10 mg·mL^-1 Tween 80+1 mg·mL^-1 TNBP,could inactivate both pseudotyped viruses after 1-hour incubation at room temperature.The titers of pseudotyped virus decreased 5.11 and 4.80 log for SARS-CoV and SARS-CoV-2 respectively.When the S/D concentration was reduced to 1%,the inactivation effect disappeared.Conclusion:Residual S/D has significant inhibitory effect on evaluating neutralizing antibody when its concentration is higher than its standard in Chinese Pharmacopoeia.To ensure the accuracy of PBNA detection,the S/D residue in plasma should not exceed the standard in Chinese Pharmacopoeia.S/D reagent has the function of inactivating SARS-CoV and SARS-CoV-2 pseudotyped viruses.
作者
王萌
张黎
李倩倩
吴佳静
郝焕
孙其玉
聂建辉
黄维金
WANG Meng;ZHANG Li;LI Qian-qian;WU Jia-jing;HAO Huan;SUN Qi-yu;NIE Jian-hui;HUANG Wei-jin(National Institutes for Food and Drug Control,Key Laboratory of the Ministry of Health for Research on Quality and Standardization of Biotech Products,Key Laboratory of Biological Product Quality Research and Evaluation of National Medical Products Administration,Beijing 102629,China)
出处
《药物分析杂志》
CAS
CSCD
北大核心
2020年第4期613-619,共7页
Chinese Journal of Pharmaceutical Analysis
基金
十三五传染病重大专项课题资助项目(2018ZX10731101-002-006,2018ZX10731101-002-009,2018ZX10731101-001-006)。
作者简介
通信作者:聂建辉,Tel:(010)53851712,E-mail:niejianhui@nifdc.org.cn;通信作者:黄维金,Tel:(010)53851737,E-mail:huangweijin@nifdc.org.cn;第一作者:王萌,Tel:(010)53851703,E-mail:william_w_m@139.com第一作者:张黎,Tel:(010)53851698,E-mail:zhangli@nifdc.org.cn。
