Indirect ELISA and Indirect Immunofluorescent Antibody Assay for Detecting the Antibody against Murine Norovirus S7 in Mice
Indirect ELISA and Indirect Immunofluorescent Antibody Assay for Detecting the Antibody against Murine Norovirus S7 in Mice
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DOI：
10.1538/expanim.59.47
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发表时间：
2010-01-01
影响因子：
2.4
通讯作者：
Yoshikawa, Yasuhiro
中科院分区：
文献类型：
--
作者：
Kitagawa, Yota;Tohya, Yukinobu;Yoshikawa, Yasuhiro
为评价小鼠诺如病毒(MNV)感染状况，建立了检测小鼠MNV-S7抗体的酶联免疫吸附试验(EL ISA)和间接免疫荧光抗体(IFA)检测方法。两种检测方法均采用日本分离的MNV-S7。将感染MNV-S7的RAW 264细胞培养上清液超速离心，制备出抗MNV-S7抗体。6周龄雌性C57BL/6JJCL小鼠经口接种MNV-S7后获得阳性血清。针对感染的RAW 264细胞的IFA能够区分阳性血清和阴性血清。用福尔马林处理的MNV-S7抗原包被的96孔酶标板进行间接酶联免疫吸附试验。在该系统中，感染后2周或以后的小鼠血清的OD值显著升高，并被判定为阳性。在BALB/cAJCL、C57BL/6JJCL、DBA/2JJCL和JCL：ICR小鼠中观察到相同水平的抗MNV-S7抗体应答，而C3H/HeJJCL小鼠在感染后4周抗体产生略低。我们还使用该ELISA系统对从日本研究机构的15个常规小鼠室获得的77份小鼠血清样本进行了评估，发现大约一半的血清样本含有MNV-S7抗体。我们发现，一些血清样本对小鼠肝炎病毒和肺支原体抗体呈阴性，而对MNV-S7抗体呈阳性。结果表明，与世界其他地区的情况一样，在日本的传统小鼠群体中，MNV感染比小鼠肝炎病毒和肺支原体等其他感染更普遍。
To evaluate murine norovirus (MNV) infection in laboratory mice, we attempted to develop an enzyme-linked immunosorbent assay (ELISA) system and an indirect immunofluorescent antibody (IFA) assay for detecting the anti-MNV-S7 antibody in mice. MNV-S7, which was isolated in Japan, was used in both assays. The antigen for ELISA was prepared by ultracentrifugation of culture supernatants of RAW 264 cells infected with MNV-S7. Positive sera were obtained from 6-week-old, female C57BL/6JJcl mice inoculated orally with MNV-S7. IFA against infected RAW 264 cells was able to discriminate positive sera from negative sera. Indirect ELISA was performed using 96-well ELISA plates coated with formalin-treated MNV-S7 antigen. In this ELISA system, mouse sera obtained 2 weeks after infection or later showed significantly high OD values and were judged positive. An equal level of anti-MNV-S7 antibody response was observed in BALB/cAJcl, C57BL/6JJcl, DBA/2JJcl, and Jcl:ICR mice; whereas, C3H/HeJJcl mice demonstrated slightly lower antibody production 4 weeks after infection. We also used this ELISA system to evaluate 77 murine serum samples obtained from 15 conventional mouse rooms in research facilities in Japan and found that approximately half of the serum samples contained antibody to MNV-S7. We found that some serum samples were negative for antibodies to mouse hepatitis virus and Mycoplasma pulmonis but positive for antibody to MNV-S7. The results suggest that the MNV infection is more prevalent than other infections such as mouse hepatitis virus and Mycoplasma pulmonis in conventional mouse colonies in Japan, as is the case in other areas of the world.