Colti病毒抗原制备、保存及其在检测患者血清Colti病毒抗体中的应用

目的 获得较高滴度和稳定的Colti病毒抗原，进一步开展Colti病毒感染的血清学诊断。方法 用C6／36细胞培养Colti病毒，于不同时间收获后进行病毒滴度测定。病毒经聚乙二醇(PEG)纯化浓缩后抗原分别保存于-20℃和4℃备用。利用该抗原采用ELISA法检测患者血清标本的Colti病毒抗体。结果 在制备Colti病毒抗原时，以细胞培养3—4周的病毒滴度最高，经PEG纯化浓缩的抗原于-20℃和4℃条件下，保存6个月，其抗原滴度仍保持在较高水平。特别是加入甘油的抗原无论在-30℃，还是在4℃下，甚至可保存2年。用这些抗原，检测疑似乙型脑炎或病毒性脑炎患者标本1141份，Colti病毒IgM抗体阳性130例，阳性率为11．4％(130／1141)，特别是检测广州市儿童医院患者标本41份，9份Colti病毒抗体阳性，阳性率为22．0％，其中5例临床诊断为病毒性脑炎。结论 为建立检测Colti病毒抗体及诊断试剂盒提供了较稳定的抗原，Colti病毒可能是引起我国夏、秋季脑炎的又一重要病原。

Preparation and storage of coltivirus antigen and its application in detection of Coltivirus antibodies in serum from patients

OBJECTIVE: To prepare purified and concentrated coltivirus high titer antigen in order to further detect antibodies against coltivirus in serum sample of patients. METHODS: The coltivirus in C6/36 cells was cultured and harvested at different time, and the titer was titrated. The virus was purified and concentrated by polyethylene glycol (PEG), and stored at -20 degrees and 4 degrees, with and without glycerol, respectively, then the titer of coltivirus antigen was tested by indirect ELISA. By using the antigen, coltivirus antibodies in serum samples from both suspected Japanese encephalitis (JE) and viral encephalitis (VE) patients were detected. RESULTS: The highest titer of coltivirus was found at 3-4 weeks of culturing. The antigen titer could be maintained at least for 6 months, especially antigen with glycerol either at 4 degrees or at -30 degrees even for two years. Totally 1141 serum samples from patients diagnosed clinically as JE and VE were tested. The results showed that 130 samples were coltivirus IgM antibody positive and the average positive rate was 11.4% (130/1141). Among 41 samples of paired-serum from patients in Guangzhou Children's Hospital, 9 samples were positive, the positive rate was 22.0% (9/41) in which 5 samples were diagnosed clinically as VE. CONCLUSIONS: Stable and purified coltivirus antigen was obtained in order to test coltivirus antibodies as well as development of kits. Coltivirus probably can cause summer-autumn encephalitis in China.