猪源脑心肌炎病毒单克隆抗体研制与免疫学特性测定

Preparation of monoclonal antibodies against encephalomyocarditis virus from swine and immunological characterization

利用纯化灭活的猪源脑心肌炎病毒(EMCV)免疫BALB/c小鼠,采用杂交瘤细胞技术,研制获得4株能稳定分泌抗EMCV抗体的杂交瘤细胞株,分别命名为1D1、2A2、2B6和4E2。经ELISA测定,细胞培养上清中抗体效价分别为1∶1 600,1∶6 400,1∶6 400和1∶3 200,小鼠腹水抗体效价分别为1∶1.63×106,1∶3.28×106,1∶1.13×106和1∶5.63×105。1D1和2A2单抗亚类为IgG1,2B6和4E2单抗亚类为IgG2b,轻链均为κ型。Western blot结果表明,1D1、2A2和4E2能特异性识别病毒的VP1蛋白,2B6能特异性识别病毒的VP2蛋白。间接免疫荧光试验证明,4株单抗具有良好的特异性,均能识别EMCV。本研究获得的4株特异性单抗,将为EMCV诊断方法的建立和病毒蛋白功能的研究奠定基础。

The purified encephalomyocarditis virus （EMCV） from swine was used to immunize BALB/c mice, and the stimulated spleno- cytes were fused with myeloma cells of SP2/0 to produce hybridomas. Four stable murine monoclonal cell lines producing antibodies （McAbs） against EMCV were generated and named as 1D1, 2A2, 2B6 and 4E2, respectively. The titers of the four McAbs in the superna- tant of cell culture were 1 ： 1 600, 1 ： 6 400, 1 ： 6 400 and 1 ： 3 200, respectively, and the titers in ascites were 1 ： 1.63×10^6, 1 ： 3.28 ×10^6 , 1 ： 1.13×10^6 and 1 ： 5.63×10^5, respectively. 1D1 and 2A2 belong to the IgG1 subtype, while 2B6 and 4E2 belong to the IgG2b subtype. All of the McAbs hadKlight chain. Western blot analysis indicated that 1 D1,2A2 and 4E2 could react with VP1 protein of EMCV, while 2B6 was able to recognize the VP2 protein of EMCV. The indirect immunofluorescence assay results showed that all of them could react with BHK-21 cells infected by EMCV, indicating that the four McAbs had good specificity and can be used for the diagnosis of EMCV. This study provides a basis for the functions of viral proteins.