小鼠诺如病毒间接免疫荧光检测方法的建立

目的建立小鼠诺如病毒(MNV)的间接免疫荧光试验(IFA)的检测方法。方法将MNV-1接种RAW264.7细胞,培养36 h收集病毒培养物及未感染细胞培养物,点制抗原玻片;以107TCID50的MNV-1灌胃接种BALB/c小鼠,收集感染血清做阳性对照血清。收集人工感染后不同时间点的血清样品,以1:10的稀释度使用IFA法测定感染血清MNV抗体滴度。选取80份送检小鼠血清样品,以IFA和ELISA方法测定,差异样品使用Western blot方法进行验证。结果 MNV-1感染RAW264.7细胞(36~48)h,细胞感染率为60%,以36 h感染细胞状态为佳;IFA法测定感染小鼠血清,小鼠感染1周后抗体水平逐渐提高,在4周时抗体滴度达到最高值,之后维持稳定,采集感染4周的动物血清做IFA方法的阳性质控品;80份血清中,IFA法测定阳性27份,阴性53份,ELISA法测定阳性32份,阴性48份;Western blot方法对差异样品验证,其中阳性3份,阴性2份,IFA法和ELISA法检测符合率分别为96.0%和97.5%。结论 IFA方法检测小鼠诺如病毒基本可以反应小鼠的感染情况,偶有假阴性的出现,可以选择IFA方法和ELISA方法作为初筛,差异样品用Western blot方法验证。

Method establishment of indirect immunofluorescence assay for Murine Norovirus

Objective To establish an indirect immunofluorescence assay for detection of murine norovirus (MNV). Methods Mouse leukaemic monocyte macrophage cell line RAW264.7 cells were infected with MNV-1 and cultured for 36 hours to collect the virus and uninfected cells, and to make antigen glass slides. BALB/c mice were gavaged with MNV-1 (10⁷TCID50) and infected sera were collected as positive control. The serum was 1:10 diluted and used for measuring MNV antibody by immunofluorescence assay (IFA). 80 serum samples were tested using the two methods, IFA and ELISA, and the discrepant samples were validated by Western blotting. Results RAW264.7 cells were infected with MNV-1 for 36-48 h, showing an infection rate of 60% of the cells, and the cells infected for 36 h were preferred. IFA method was used to detect the serum with MNV-1 infection and showed that the antibody content was gradually increased at one week after infection, reaching a maximum antibody concentration at 4 weeks after infection, and maintained a stable level later. The mouse serum at four weeks after MNV-1infection was used as positive quality control. Among the 80 serum samples, 27 positive and 53 negative cases were detected by IFA method, and 32 positive and 48 negative cases were detected by ELISA. The five discrepant samples were verified by Western blotting, resulted in 3 positive and 2 negative cases. The coincidence rate of IFA was 96.0% and that of ELISA methods was 97.5%. Conclusions Basically, immunofluorescence assay can be used to detect the MNV-1 infection in mice, although false negative result may occur occasionally. IFA and ELISA detection can be selected as initial screening measures, and use Western blot assay to verify the discrepant samples.