柯萨奇病毒A16型快速纯化和中和性单克隆抗体制备与鉴定

Purification of coxsackievirus A16 viral particles and preparation and identification of neutralizing monoclonal antibody against coxsackievirus A16

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10^3,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 the rapid purification of Coxsackievirus A16 using ultracentrifugation.And To prepare and identify the neutralizing monoclonal antibody against CA16.Methods The CA16 culture supernatant was harvested and then concentrated by 100 Kcapsule.The concentration of CA16 was purified by cesium chloride ultracentrifugation.Purification of CA16 were identified by transmission electron microscopy.BALB/c mice were immunized with inactivated CA16.Spleen cells were harvested and fused with SP2/0myeloma cells,hybridoma cell strain secreting mAb against CA16 were objected to screening.Characterization of the prepared mAb were analyzed by ELISA and microneutralization assay.Results The purified CA16 method of cesium chloride gradient ultracentrifugation was established,TEM analysis was showed that CA16 particles have icosahedral structure,the diameters of the viral particles were approximately 20-30 nm.Two hybridoma cell strains secreting mAb against CA16 were obtained,the subtypes of two mAbs were IgG2 a,the binding titers of Anti/CA16/5and Anti/CA16/10 were 10^3 and 104 respectively.Neutralizing titer of the two mAbs were 1∶256and 1∶1 024 respectively.Conclusion Establishment method of cesium chloride gradient ultracentrifugation was performed to purify CA16,the two mAbs with neutralizing ability to against CA16 may become application of treatment and vaccine.