呼肠孤病毒Ⅲ型免疫荧光检测方法的建立及初步应用

Establishment and Initial Application of an Immunofluorescence Assay in Detecting Mammalian Orthoreovirus 3

目的 建立呼肠孤病毒Ⅲ型(Reo3)免疫荧光(IFA)检测方法,应用于人用动物源性生物材料及生物制品外源Reo3的检测。方法滴定病毒TCID50,筛选Reo3敏感细胞,依据国标IFA法制备抗原片,方阵法滴定免疫荧光素最佳工作浓度。并进行特异性、敏感性和稳定性试验。结果选取BSC-1细胞作为Reo3敏感细胞,病毒感染力滴度(TCID50)为10-5.8/mL;免疫荧光素最佳工作浓度为1∶100;与小鼠鼠痘(Ect)病毒、小鼠肝炎(MHV)病毒均无交叉反应;稳定性和敏感性试验显示,不同时间IFA检测灵敏度均为1∶1280;可检测到的病毒滴度最低为10-4.1/mL。结论建立的IFA法敏感性、特异性强,稳定性好,可用于人用动物源性生物材料及生物制品Reo3的检测。

Objective To establish an immunofluorescence assay（IFA） method of detecting the mammalian orthoreovirus 3（Reo3） in animal-derived biological materials and biological products for human use.Methods Reo3-sensitive cells were screened.The virus TCID50 was titrated and antigen slides were prepared according to GB IFA.The best working concentration of IgG-FITC was titrated and the specificity,sensitivity and stability were tested.Results BSC-1 cells were selected as Japanese encephalitis virus（JEV）-sensitive cells.The TCID50 of the virus was 10-5.8·mL-1.The best working concentration of the IgG-FITC was 1：100.There was no cross-reactivity with ectromelia virus and mouse hepatitis virus.The tests of sensitivity and stability showed that the detection sensitivity was 1：1280 at different time points.The lowest detected virus titer was 10-4.1·mL-1.Conclusions The established IFA method is good in sensitivity,specificity and stability,and can be used in detecting the Reo3 in animal-derived biological materials and biological products for human use.