小鼠血清乙型肝炎病毒前S2抗体间接ELISA检测方法的建立

Development of an indirect ELISA method for hepatitis B virus PreS2 antibody in mouse serum

目的建立小鼠血清乙型肝炎病毒(Hepatitis B virus,HBV)前S2抗体间接ELISA检测方法。方法以HBV前S2(1～26)多肽包被酶标板,利用方阵滴定法确定间接ELISA法的最适工作条件,验证该方法的精密度、灵敏度和特异性,并与商品化试剂盒进行比较。结果确定最佳抗原包被浓度为2μg/ml;血清稀释度为1︰10,37℃反应30 min;HRP标记的羊抗小鼠IgG最佳稀释度为1︰10 000,37℃反应30 min;TMB底物37℃显色15 min,以2.1×阴性对照血清A450均值(阴性对照血清小于0.05时按0.05计算)为Cut-off值。该方法具有良好的试验内和试验间精密度;与商品化试剂盒比较,灵敏度为100%,特异性为97.3%,二者符合率为98.5%。结论已成功建立HBV前S2抗体间接ELISA方法,可用于小鼠血清中前S2抗体的检测。

Objective To develop an indirect ELISA method for hepatitis B virus（ HBV ） PreS2 antibody in mouse serum. Methods Microtiter wells were coated with HBV PreS2 polypeptides （1-26）, based on which the reaction condition of indirect ELISA was optimized by block titration. The developed method was verified for precision, sensitivity and specificity, and the results were compared with those by commercial kit. Results The optimal antigen concentration for coating was 2μg/ml, and the optimal dilution of serum was 1 ： 10, while the optimal reaction temperature and time of serum were 37 ℃ and 30 min respectively. However, the optimal dilution reaction temperature and time of HRPlabeled goat anti-mouse IgG were 1： 10 000, 37 ℃ and 30 min respectively. The optimal temperature and time for coloration of TMB substrate were 37 ℃ and 15 min respectively. The 2. 1 folds of A450 value of negative control serum was served as cut-off value. The A450 value of negative control serum less than 0. 05 was calculated as 0. 05. The developed method showed high intra- and inter-precisions, of which the sensitivity and specificity were 100% and 97. 3% respectively. The coincidence rate of detection results by the developed method and the commercial kit was 98. 5%. Conclusion An indirect ELISA method suitable for detection of HBV PreS2 antibody in mouse serum was successfully developed.