摘要
目的制备小鼠脑脊髓炎病毒（TMEV）标准血清，建立ELISA检测方法，实现一种病毒多种检测方法的比对研究。方法用TMEV感染3周龄SPF级BALB／c小鼠，制备免疫用抗原。分别在0、7、14d以腹腔注射的方式免疫SPF级6-8周龄BALB／c小鼠，免疫血清用IFA、IEA法进行鉴定；TMEV感染BHK-21细胞，制备EHSA抗原，确定酶结合物、抗原和标准阳性血清的最佳工作浓度，建立TMEVEHSA检测方法，进行敏感性、特异性、重复性和稳定性实验。结果制备TMEV免疫血清，以IFA、IEA测定血清效价分别为1：640和1：320，与痘苗病毒、小鼠肺炎病毒、小鼠肝炎病毒、仙台病毒和呼肠孤病毒-Ⅲ型均无交叉反应；建立了ELISA检测方法，确立了各种试剂的最佳工作浓度，其中酶结合物最佳工作浓度为1：10000，抗原为2.5μg／mL，阳性参考血清为1：200；EHSA检测灵敏度为1：3200。板内特异抗原、正常抗原平均变异系数为4.67％和5.7％，板间为4.39％和7.61％。稳定性实验相对偏差均小于20％。结论本研究制备的TMEV免疫血清可作为血清学检测方法中的标准质控血清；建立的ELISA方法重复性、稳定性好，敏感性和特异性强，可用于小鼠脑脊髓炎病毒血清抗体检测。
Objective To prepare the standard serum against Theiler＇s mouse encephalomyelitis virus （TMEV） and establish a method of detecting TMEV antibody by ELISA, for viral detection by cross-reference multi-method in laboratory animals. Methods TMEV was cultivated by intracerebral inoculation in BALB/c mice and immunogen was prepared. The BALB/ c mice were immunized on day 0,7,14, respectively, by intraperitoneal injection of the immunogen. The harvested sera were verified for the sensitivity with IFA and IEA. BHK-21 cells were infected with TMEV to prepare specific TMEV antigen, while the normal BHK-21 ceils as control antigen. To titrate the best working concentration of enzyme conjugate, antigen and the positive serum. To establish the TMEV ELISA detection technique, and the sensitivity, specificity, repeatability and stability were determined. Results The sera titer was 1 ： 640 determined by IFA and 1 ： 320 by IEA. The best working concentration of enzyme cojugate,antigen and positive serum were 1 ： 10000,2.5 ttg/mL,and 1：200, respectively. The ELISA detection sensitivity was 1： 3200. There was no cross-reactivity with Pneumonia virus of mice （PVM）, mouse hepatitis virus （MHV）, Sendai virus （SV） and Reovirus-3 （Reo-3）. The inter-assay coefficients of variation of specific antigen and normal antigen were 4.67 % and 5.7 %, and inter-plate coefficients of variation of specific antigen and normal antigen were 4.39 % and 7.61%, respectively. The stability test showed a relative deviation below 20%. Conclusion The immunized serum could be used as the standard control serum in TMEV serological detection methods. The ELISA method has good sensitivity, specificity, repeatability and stability. Therefore, this ELISA technique can be used in serological detection of Theiler＇s mouse encephalomyelitis virus.
出处
《中国比较医学杂志》
CAS
2009年第12期5-9,共5页
Chinese Journal of Comparative Medicine
基金
中央级公益性科研院所基本科研业务费专项资金
项目号:DWS200708
科技部基础专项重点项目
项目号:2002DEA20023
科技部条件基础平台专项重点项目
项目号:2003DIA7J033
