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目的建立柯萨奇病毒A16型快速纯化方法,制备中和性单抗,并对单抗进行分析。方法收获CA16培养上清液,超滤浓缩,氯化铯密度梯度离心纯化病毒颗粒,透射电镜鉴定纯化产物。福尔马林灭活CA16,免疫BALB/c小鼠,制备分泌抗CA16特异性单抗的杂交瘤细胞系,用ELISA和中和试验分别对单抗特性进行分析。结果初步建立CA16病毒氯化铯密度梯度纯化方法,电镜显示,病毒颗粒为二十面体立体对称球形结构,病毒直径在20~30nm间,大小均匀。获得2株分泌抗CA16单抗的杂交瘤细胞系,2株单抗均为IgG2a亚型,Anti/CA16/5效价为103,Anti/CA16/10效价为104。2株抗体的中和效价分别为1∶256和1∶1 024。结论初步建立氯化铯密度梯度纯化CA16的方法,筛选出2株具有中和活性的抗CA16单抗,为CA16病毒的基础研究提供重要的原材料。
Objective To establish a rapid purification method of coxsackie virus A16, prepare neutralizing McAb, and analyze the monoclonal antibody. Methods The supernatant of CA16 was harvested, concentrated by ultrafiltration and purified by cesium chloride density gradient centrifugation. The purified product was identified by transmission electron microscopy. Formalin was used to inactivate CA16 and BALB / c mice were immunized to prepare hybridoma cell lines secreting anti-CA16 specific mAb. The characteristics of mAb were analyzed by ELISA and neutralization assays respectively. Results The method of density gradient purification of cesium chloride of CA16 was initially established. Electron microscopy showed that the virus particle was an icosahedral symmetrical spherical structure with a diameter of 20-30 nm and a uniform size. Two hybridoma cell lines secreting anti-CA16 McAb were obtained. The two McAbs were all IgG2a subtype, the titer of Anti / CA16 / 5 was 103 and the neutralization titer of Anti / CA16 / 10 was 104.2 The titers were 1:256 and 1: 1 024, respectively. Conclusion The method of purifying CA16 by density gradient of cesium chloride was established. Two anti-CA16 monoclonal antibodies with neutralizing activity were screened out, which provided important raw materials for the basic research of CA16 virus.