戊型肝炎病毒重组蛋白P179抗原单克隆抗体及抗原检测试剂盒的制备

Preparation of monoclonal antibody against recombinant protein P179 of hepatitis E virus and detection kit for P179

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 the monoclonal antibody（McAb）against recombinant protein 179 of hepatitis E virus（HEV）and the detection kit for P179.Methods BALB / c mice were immunized with recombinant protein P179 of ORF2 of HEV type 4,of which the splenic cells were fused with SP2 / 0 cells.Positive hybridoma cell strains were screened by indirect ELISA,subjected to cloning culture by limited dilution and prepared into ascites,then counted for chromosome,and analyzed for the subclass,specificity and stability of secreted McAb.The purity of McAb was determined by SDS-PAGE,based on which a double antibody sandwich ELISA kit was prepared using rabbit anti-P179 polyclonal antibody as coating antibody and HRP-labeled McAb against P179 as enzyme labeled antibody,determined for linear range,and verified for accuracy,precision and stability.Results Four cell strains stably secreting McAbs against recombinant protein P179,named as 3A10,3F8,4E9 and 5G6,were obtained,in which the numbers of the chromosomes were 96,98,101,99,respectively.The McAbs secreted by the four cell strains were of subclasses IgG2a,IgG2a,IgG1 and IgG2b,of which the titers were 1 ∶ 10-7、1 ∶ 10-6,1 ∶ 10-6 and 1 ∶ 10-7 respectively.After the four cell strains were subcultured continuously for 3 months and stored in frozen in liquid nitrogen for 12 months,no significant changes were observed in the titers of secreted McAbs.The prepared ELISA kit showed high linearity（R2 〉 0.950 0）,accuracy and precision,of which the intra-CV value and recovery rate were 4.17% ～ 6.26% and 87.9% ～ 114.8%,and the inter-CV value and recovery rate were 5.82% ～ 8.01% and 90.8% ～ 108.9%,respectively.The kit showed high stability after storage at 37 and 4 ℃ for 3 d.Conclusion The McAbs against recombinant protein P179 of HEV as well as the detection kit for P179 antigen were successfully prepared,which might be used for quantitative detection of vaccine antigen during production of HE vaccine.