摘要
目的制备鼠源戊型肝炎病毒（Hepatitis E Virus,HEV）抗体定量参考品,建立小鼠HEV IgG抗体定量检测方法,并对其进行验证及初步应用。方法制备鼠源抗-HEV IgG参考血清MS1,以世界卫生组织人源抗-HEV Ig标准品（NIBSC code：95/584）对其进行标定并检测其稳定性;以标定的参考品为标准,建立小鼠HEV IgG抗体定量检测方法,对线性、范围、重复性、准确度等进行验证,并对戊肝疫苗免疫后小鼠血清进行HEV IgG抗体定量检测。结果制备的MS1血清含量为30.9U/m L（95%CI：±1.1）,CV为4.8%;加速稳定性试验和冻融稳定性试验中,MS1含量CV均〈15%。建立的小鼠抗-HEV IgG抗体定量检测方法在0.01~0.15 U/m L范围内具有良好的线性（r〉0.99）;灵敏度为0.007 U/m L;对高、中、低3份不同含量抗-HEV阳性小鼠血清重复检测3次,CV均〈10%;加样回收率为83.8%~107.7%。戊肝疫苗免疫1 w,3 w,5 w时,小鼠血清HEV IgG抗体均值分别为0.3 U/m L、39.4 U/m L、345.4 U/m L。戊肝疫苗初次免疫小鼠1 w后抗体阳转率为100%,但抗体均处于较低水平;血清抗体水平随着免疫针次的增加而升高（P〈0.05）。结论制备的鼠源HEV IgG抗体定量参考稳定性良好的,建立的小鼠HEV IgG抗体定量检测方法,具有良好的灵敏度、重复性及准确度,可用于小鼠实验中戊肝疫苗免疫原性的评价。
Objective To prepare a reference in quantitative analysis of mouse anti-HEV serum IgG and develop a quantitative assay in detection of mouse anti-HEV IgG antibodies,and their＇s verification and preliminary application tests were carried out. Methods The mouse anti-HEV sera were pooled together and designated as MS1,according to the requirements for establishing antibody reference,MS1 was first calibrated based on the standard（ NIBSC code： 95 /584） from WHO,then analyzed with stability test. The calibrated refrence was used as a standard,and set up a quantitative assay in detection of mouse anti-HEV IgG antibodies,the linearity,range,repeatability,and accuracy for the assay were verified. Finally,serum samples from mice vaccinated by HE vaccine were in a quantitative analysis. Results The anti-HEV antibody content for MS1 was determined as 30. 9 U / m L（ 95% CI： ±1. 1）,and with a coefficient variation（ CV） 4. 8%. All CV for MS1 contents were 15% in accelerated steability test and freezing-thowing steability test. The assay had a good linearity（ r0. 99） in a range 0. 01 ~ 0. 15 U/m L,the sensitivity was 0. 007 U/m L. All CV were 10% in detection samples with high,medium and low level mouse antibodies,the assays were repeatly performed 3 times,and recovery rate was in 83. 8%-107. 7%. The average level of anti-HEV mouse antibody was 0. 3 U / m L,39. 4 U / m L,and 345. 4 U / m L respectivelly,after vaccinated HEV vaccine in 1,3,and 3 weeks. The antibody was in a complete converted rate of 100% but in presence at low level,after the preliminary vaccination. The antuibody level was increased（ P0. 05） by a continual immunization. Conclusion The value of MS1 reference for mouse anti-HEV antibody and an ELISA based on mouse anti-HEV quantitation quantification were successfully set up,and with advantagies as good sensitivity,repeatability and accuracy. This work will facilitate immunogenicity evaluation of HEV vaccines in mouse model.
出处
《微生物学免疫学进展》
2016年第4期17-20,共4页
Progress In Microbiology and Immunology
基金
国家"十二五"科技重大专项课题(2012ZX10004701)
关键词
戊型肝炎病毒
抗体
定量
参考品
检测方法
Hepatitis E virus（HEV）
Antibody
Quantitation
Reference
Assay
