摘要
目的建立呼吸道合胞病毒(respiratory syncytial virus,RSV)滴度的间接免疫荧光检测方法,并进行验证。方法以Synagis(Palivizumab)为一抗,荧光标记羊抗人IgG为二抗,建立用于RSV滴度检测的间接免疫荧光法,并对HEp-2细胞接种密度(3×104、4×104、5×104、6×104、7×104、8×104个/孔)、细胞培养液中胎牛血清浓度(2.5%、5%、10%、20%)、病毒培养时间(20、24、26、30、38、48 h)、抗体稀释度[一抗:1∶(1000/3)、1∶1000、1∶3000、1∶9000,二抗:1∶(400/3)、1∶400、1∶1200]进行优化。验证方法的特异性、线性范围、准确性、重复性。结果建立的方法最佳工作条件:HEp-2细胞接种密度为6×104个/孔,胎牛血清浓度为2.5%,病毒培养时间为24~26 h,一抗稀释度为1∶1000,二抗稀释度为1∶400。RSV可见特异性荧光,狂犬病病毒、流感病毒、风疹病毒均未见荧光;RSV滴度理论值在1.70×107~5.43×103 FFU/mL范围内,与检测值呈良好的线性关系,回归方程为y=1.0313 x-0.2508,R2=0.9897;准确性验证回收率在70%~130%范围内;重复性验证RSD均≤15%。结论建立的RSV滴度间接免疫荧光检测方法具有良好的特异性、准确性、重复性,且检测周期短,可用于RSV滴度快速检测。
Objective To develop and verify an indirect immunofluorescence assay(IFA)for respiratory syncytial virus(RSV)titer.Methods An indirect IFA for RSV titer was developed using Synagis(Palivizumab)as the first antibody while the fluorescence-labeled goat anti-human IgG as the second antibody.The density of HEp-2 cells inoculated(3×104,4×104,5×104,6×104,7×104 and 8×104 cells/well),fetal bovine serum(FBS)concentration in medium(2.5%,5%,10%and 20%),virus culture time(20,24,26,30,38 and 48 h)and dilutions for antibodies[1∶(1000/3),1∶1000,1∶3000 and 1∶9000 for the first antibody;1∶(400/3),1∶400 and 1∶1200 for the second antibody]were optimized.The developed method was verified for specificity,linear range,accuracy and reproducibility.Results The optimal density of HEp-2 cells inoculated,FBS concentration,virus culture time and the dilutions of the first and the second antibodies were 6×104 cells/well,2.5%,24~26 h,1∶1000 and 1∶400 respectively.Specific fluorescence was observed only in RSV while not in rabies,influenza and rubella viruses.The RSV titer in theory was 1.70×107~5.43×103 FFU/mL,which showed good linear relationship to the measured values,with a regression equation of y=1.0313 x-0.2508,R2=0.9897.The recovery rate in verification for accuracy was 70%~130%.All the RSDs in verification for reproducibility was not more than 15%.Conclusion An indirect IFA for RSV titer was developed,which showed good specificity,accuracy and precision and was time-saving,and might be used for rapid determination of RSV titer.
作者
宋月爽
陈晓旭
潘东
卢井才
张立娜
刘禹壮
原秀娟
段盛博
袁若森
赵慧
SONG Yue-shuang;CHEN Xiao-xu;PAN Dong;LU Jing-cai;ZHANG Li-na;LIU Yu-zhuang;YUAN Xiu-juan;DUAN Sheng-bo;YUAN Ruo-sen;ZHAO Hui(Changchun BCHT Biopharm Co,Ltd,Changchun 130012,Jilin Province,China;不详)
出处
《中国生物制品学杂志》
CAS
CSCD
北大核心
2022年第8期1002-1005,1012,共5页
Chinese Journal of Biologicals
基金
吉林省科技发展计划项目(20200404119YY).
关键词
呼吸道合胞病毒
间接免疫荧光法
病毒滴度
Respiratory syncytial virus(RSV)
Indirect immunofluorescence assay
Virus titer
