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目的制备戊型肝炎病毒(Hepatitis E virus,HEV)重组蛋白P179抗原单克隆抗体及抗原检测试剂盒。方法以4型HEV ORF2重组蛋白P179免疫BALB/c小鼠,取免疫小鼠脾细胞与SP2/0骨髓瘤细胞进行融合,间接ELISA法筛选阳性杂交瘤细胞株,有限稀释法进行克隆化培养,制备腹水,并对杂交瘤细胞进行染色体计数,鉴定单抗亚类及特异性、并检测稳定性;经SDS-PAGE分析抗体纯度,以兔抗179抗原多抗作为包被抗体,HRP标记P179单抗作为酶标抗体,建立双抗体夹心ELISA法,制备试剂盒,并进行最佳线性范围测定、准确性、精密性和稳定性验证。结果获得4株能稳定分泌抗P179抗原单抗的杂交瘤细胞株,分别命名为3A10、3F8、4E9和5G6,腹水抗体效价分别为1∶10-7、1∶10-6、1∶10-6和1∶10-7,染色体数分别为96、98、101和99条;亚类分别为IgG2a、IgG2a、IgG1和IgG2b;4株单抗连续传代3个月,液氮冻存1年抗体效价未发生变化;制备的试剂盒有良好的线性(R2>0.950 0)、准确性、精密性,试验内变异系数为4.17%～6.26%,回收率在87.9%～114.8%之间,试验间变异系数为5.82%～8.01%,回收率在90.8%～108.9%之间;于37℃及4℃放置3 d,仍具有良好的稳定性。结论成功制备了戊型肝炎病毒P179抗原单克隆抗体及抗原检测试剂盒,可用于戊型肝炎疫苗生产中定量检测疫苗抗原。
Objective To prepare a monoclonal antibody against Hepatitis E virus (HEV) recombinant protein P179 and an antigen detection kit. Methods BALB / c mice were immunized with recombinant protein P179 of type 4 HEV ORF2. The spleen cells of immunized mice were fused with SP2 / 0 myeloma cells. The positive hybridoma cells were screened by indirect ELISA and cloned by limiting dilution. The ascites was prepared, and the hybridoma cells were counted for chromosome count to identify the subtype and specificity of the monoclonal antibody. The purity of the antibody was detected by SDS-PAGE. The anti-179 antigen polyclonal antibody was used as the coating antibody. HRP-labeled P179 single Antibodies as enzyme-linked antibody, the establishment of double antibody sandwich ELISA method, preparation kit, and the best linear range determination, accuracy, precision and stability verification. Results Four hybridoma cell lines that could stably secrete anti-P179 mAb were obtained and named as 3A10, 3F8, 4E9 and 5G6 respectively. The antibody titer of ascites was 1:10-7, 1:10-6 and 1:10 -6 and 1:10-7, respectively. The numbers of chromosomes were 96, 98, 101 and 99 respectively. The subclasses were IgG2a, IgG2a, IgG1 and IgG2b respectively. The four McAbs were passaged continuously for 3 months, The antibody titer did not change. The prepared kit had good linearity (R2> 0.950 0), accuracy and precision. The coefficient of variation (CV) was 4.17% ~ 6.26% and the recoveries ranged from 87.9% to 114.8% The coefficient of variation (CV) was between 5.82% and 8.01% and the recoveries ranged between 90.8% and 108.9%. The stability was still good at 37 ℃ and 4 ℃ for 3 days. Conclusion The monoclonal antibody against hepatitis E virus P179 and antigen detection kit were successfully prepared and could be used for the quantitative detection of vaccine antigen in the production of hepatitis E vaccine.