小反刍兽疫病毒核蛋白单克隆抗体的制备与初步应用

针对小反刍兽疫病毒核蛋白制备特异性的单克隆抗体,并对其进行生物学特性鉴定和初步应用。以纯化的Bacmid-PPRV-N重组蛋白为抗原免疫BALB/c小鼠,取免疫小鼠的致敏脾细胞与SP2/0骨髓瘤细胞在PEG作用下融合,获得单克隆抗体,并通过染色体技术等方法研究其生物学特性,将其作为竞争单抗,Bacmid-PPRV-N重组蛋白作为检测抗原建立竞争ELISA检测方法。结果表明:经克隆和间接ELISA筛选,获得了2株能稳定分泌抗小反刍兽疫病毒N蛋白抗体的杂交瘤细胞株,分别命名为5B11和3H10-3B8。生物学特性鉴定试验表明:5B11和3H10-3B8抗体类型和亚类均为IgG2b;5B11单抗腹水的效价达1∶819 200,3H10-3B8达1∶12 800;血清学试验证明2株单抗均能与Bacmid-PPRV-N重组蛋白抗原结合,具有高度的特异性;相加ELISA试验结果显示,5B11和3H10-3B8 2株单克隆抗体分别识别N蛋白上不同的抗原位点;2株杂交瘤细胞的染色体均为99～104。应用建立的c-ELISA检测方法对222份血清样品进行PPRV抗体的检测,与参考试剂盒比较得到98.20%的符合率。本研究获得了2株能稳定分泌抗PPRV N蛋白单克隆抗体的杂交瘤细胞株,以单抗5B11作为竞争抗体建立了PPRV的c-ELISA检测方法。

Preparation and Preliminary Application of Monoclonal Antibodies against Nucleoprotein of Peste des Petits Ruminants Virus

The objective of this study was to prepare specific monoclonal antibodies(mAbs) against nucleoprotein of peste des petits ruminants virus(PPRV),characterize its properties,and use it in ELISA.After immunization of BALB/c mice with purified recombinant protein Bacmid-PPRV-N,two monoclonal antibodies against PPRV nucleoprotein were successfully prepared by polyethyleneglycol(PEG)-mediated fusion of sensitized lymphocytes and myeloma cells and named 5B11 and 3H10-3B8,respectively.The specificity and biological characterization of the mAbs were identified.A competitive ELISA using PPRV recombinant nucleoprotein as coating antigen and mAb 5B11 as competitive antibody was established.Our results about isotypes and subclasses indicate that 5B11 and 3H10-3B8 belong to IgG2b,the titres of ascitic fluids of 5B11 was up to 1:819 200 and 3H10-3B8 was 1:12 800.These mAbs could specifically recognize recombinant protein Bacmid-PPRV-N antigens in a serologic test.Antibodies additivity assay demonstrated that 5B11 and 3H10-3B8 recognized the different epitopes of PPRV nucleoprotein.The number of chromosome of the hybridoma cell lines was 99 to 104.A total of 222 serum samples were detected in parallel by c-ELISA and reference c-ELISA kit.The coincidence rate of the two assaya was 98.15%.We successfully prepared two specific monoclonal antibodies against PPRV nucleoprotein and established the competitive immunofluorescent method for detecting PPRV.