基于N蛋白单克隆抗体的小反刍兽疫病毒抗体检测胶体金试纸条的研制

Preparation of colloidal gold test strips for the detection of antibodies to peste des petits ruminants based on monoclonal antibodies to N protein

本研究旨在建立一种简便、快捷、可直观检测小反刍兽疫病毒(peste des petits ruminants virus,PPRV)抗体的检测方法。将pET-32a-N重组质粒转化至大肠杆菌(Escherichia coli)Rosetta(DE3)感受态细胞中进行诱导表达,以纯化的PPRVN蛋白免疫8周龄BALB/c小鼠,取其脾细胞与SP2/0骨髓瘤细胞进行融合,间接酶联免疫吸附试验(enzyme-linked immunosorbent assays,ELISA)筛选及亚克隆,获得了抗PPRV N蛋白的单克隆抗体。将PPRV N蛋白分别作为金标抗原及检测线(T线)包被抗原、单克隆抗体作为质控线(C线)包被抗体,组装成检测PPRVN蛋白抗体的胶体金免疫层析试纸条。结果显示:成功获得1株能稳定分泌抗N蛋白抗体的杂交瘤细胞株,命名为1F1;间接ELISA检测1F1腹水效价为1:128000;亚类鉴定结果为IgG1,轻链为kappa链。Westernblotting结果显示,1F1能与PPRV N蛋白特异性结合;间接免疫荧光(indirect immunofluorescent assay,IFA)结果显示,制备的单克隆抗体能够识别PPRV;建立的试纸条能够特异性地检测PPRV抗体,以不同批次的试纸条重复检测,结果无差异。根据122份临床血清的检测结果,PPRV抗体试纸条与ELISA试验的符合率为97.6%。本研究建立的试纸条检测方法具有良好的特异性、重复性和敏感性,可用于PPRV抗体的快速检测。

A simple,fast,and visual method for detecting antibodies against peste des petits ruminants virus(PPRV)using colloidal gold strips was developed.In this study,the pET-32a-N was transformed into Escherichia coli Rosetta(DE3)for expression.Hybridoma cell lines were generated by fusing SP2/0 myeloma cells with splenocytes from immunized mice with the expressed and purified N protein of PPRV.The PPRV N protein was labeled with colloidal gold particles as the gold-labeled antigen.The N protein served as the gold standard antigen and as the test(T)line-coated antigen,while the monoclonal antibody served as the quality control(C)line-coated antibody to assemble the colloidal gold immunochromatographic test strips for detecting antibodies against the N protein of PPRV.Hybridoma cell line designated as 1F1 was able to stably secrete the monoclonal antibody against the N protein of PPRV.The titer of 1F1 monoclonal antibody in ascites was 1:128000 determined by indirect enzyme-linked immunosorbent assays(ELISA),and the immunoglobulin subtype of the monoclonal antibody was IgG1,with kappa chain.The obtained monoclonal antibody was able to specifically recognize the N protein of PPRV,as shown by Western blotting and indirect immunofluorescent assay(IFA).The developed colloidal gold test strip method was able to detect PPRV antibodies specifically,and there was no difference between different batches of the test strips.Testing of a total of 122 clinical sera showed that the compliance rate of the test strip with ELISA test was 97.6%.The test strip assay developed in this study has good specificity,reproducibility,and sensitivity,and it can be used for the rapid detection of PPRV antibodies.