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目的建立重组戊型肝炎p179疫苗抗体间接ELISA检测方法,并进行初步应用。方法确定重组戊型肝炎疫苗p179抗原的最佳包被浓度及包被条件、酶标抗体的最佳稀释度、封闭条件、最佳抗体稀释液,对建立的方法的特异性、准确性及精密度进行验证,并与市售ELISA试剂盒进行比较。结果抗原最佳包被浓度为1μg/ml,4℃包被过夜;酶标抗体最佳稀释度为1∶5 000;3%牛血清白蛋白封闭3 h;最佳抗体稀释液为含1%酪蛋白PBS。建立的方法检测的A450与重组戊肝抗原浓度呈明显的剂量依赖性,对不相关的anti-HAV、anti-HB、anti-INF、anti-RABV无交叉反应;36份小鼠阳性血清的检出率为100%;3份阳性血清重复检测20次的CV均小于15%。市售某厂家ELISA试剂盒检测48份血清,阳性检出率为83.3%,建立的间接ELISA方法检测,阳性检出率为89.6%。结论建立了重组戊肝p179疫苗抗体间接ELISA检测方法,可用于重组戊肝疫苗小鼠免疫后血清抗体的检测。
Objective To establish an indirect ELISA for the detection of recombinant hepatitis E virus vaccine p179 and to carry out preliminary application. Methods The optimal concentration and coating conditions of recombinant hepatitis E vaccine p179 antigen, the optimal dilution of enzyme-labeled antibody, the blocking conditions and the optimal antibody dilution were determined. The specificity, accuracy and precision of the established method Degree of validation and comparison with commercially available ELISA kits. Results The optimum antigen concentration was 1μg / ml and was coated overnight at 4 ℃. The best dilution of enzyme-labeled antibody was 1: 5000 and 3% of BSA for 3 h. The best antibody dilution was 1% Casein PBS. The established method of detecting A450 and recombinant hepatitis E antigen concentration was significantly dose-dependent, no correlation of anti-HAV, anti-HB, anti-INF, anti-RABV no cross-reaction; 36 positive sera The rate of 100%; 3 positive serum 20 times repeated detection of CV were less than 15%. A commercial ELISA kit was used to detect 48 sera. The positive detection rate was 83.3%. The established indirect ELISA method was used to detect the positive rate of 89.6%. Conclusion The indirect ELISA method for the detection of recombinant hepatitis E virus vaccine p179 was established and could be used to detect the serum antibody after immunization with recombinant hepatitis E vaccine.