CAJ | 학술논문

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107 TCID50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。
목적：건립소서낙여병독（ MNV）적간접면역형광시험（ IFA）적검측방법。방법장MNV-1접충RAW264.7세포，배양36 h수집병독배양물급미감염세포배양물，점제항원파편；이107 TCID50적MNV-1관위접충BALB／c소서，수집감염혈청주양성대조혈청。수집인공감염후불동시간점적혈청양품，이1：10적희석도사용IFA법측정감염혈청MNV항체적도。선취80빈송검소서혈청양품，이IFA화ELISA방법측정，차이양품사용Western blot방법진행험증。결과 MNV-1감염RAW264.7세포（36～48）h，세포감염솔위60％，이36 h감염세포상태위가；IFA법측정감염소서혈청，소서감염1주후항체수평축점제고，재4주시항체적도체도최고치，지후유지은정，채집감염4주적동물혈청주IFA방법적양성질공품；80빈혈청중，IFA법측정양성27빈，음성53빈，ELISA법측정양성32빈，음성48빈；Western blot방법대차이양품험증，기중양성3빈，음성2빈，IFA법화ELISA법검측부합솔분별위96.0％화97.5％。결론 IFA방법검측소서낙여병독기본가이반응소서적감염정황，우유가음성적출현，가이선택IFA방법화ELISA방법작위초사，차이양품용Western blot방법험증。
Objective To establish an indirect immunofluorescence assay for detection of murine norovirus ( MNV) .Methods Mouse leukaemic monocyte macrophage cell line RAW 264.7 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 (107 TCID50) 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 .