Liu Yang, Zhao Xiangrong, Guo Chunyan, Li Yan, Wang Guanghua, Liang Daoyan, Yu Pengbo, Li Yuan, Hu Jun
Central Laboratory, Shaanxi Provincial People's Hospital, Third Affiliated Hospital, Medical College, Xi'an Jiaotong University, Xi'an 710068, China.
Shaanxi Provincial Centre for Disease Control and Prevention, Xi'an 710052, China.
Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi. 2017 May;33(5):688-694.
Objective To prepare the monoclonal antibody (mAb) against influenza A virus (H1N1) using purified viral particles as antigen and investigate the characterization of host cells infected with influenza virus utilizing the mAb. Methods A/PR/8 (H1N1) virus was cultured in embryonated chicken eggs and further purified by differential and density gradient centrifugation. The structure of viral particles was identified by transmission electron microcopy (TEM). Immunogenicity of purified virus was evaluated by Balb/c mice immunized with formalin-inactivated virus. Hybridomas secreting mAbs were established through a fusion of Sp2/0 myeloma cells and splenocytes from the mice immunized with the virus. The characteristics of mAb were identified by ELISA, immunofluorescence assay (IFA), Western blotting, hemagglutinin inhibition assay (HI) and microneutralization assay. The outside hemagglutinin (HA) on the plasma membrane of the host MDCK cells in which the viruses were propagated and the apoptosis of MDCK cells infected with the viruses were measured using flow cytometry. Cell-based ELISA was established using mAb specific to HA. Subsequently, the growth of virus was analyzed by cell-based ELISA. Results Transmission electron microscopy revealed that the physical structures of the purified virus were spherical, elliptical and extended threadlike. Serum IgG titer to influenza virus showed a progressive increase, and the IgG titer reached 10 after the immunization for 6 weeks. Six hybridoma clones secreting mAb specific to A/PR/8 were developed by hybridoma technology. The HI and neutralization activities of PR8-10 mAb were significantly higher than those of the other mAbs. HI and neutralization titers of PR8-10 mAb were 1:2048 and 1:640, respectively. IFA and Western blotting confirmed that PR8-10 mAb could recognize HA. Flow cytometry showed that PR8-10 mAb also recognized HA on the membrane of MDCK in which the viruses were replicated and virus infection induced the apoptosis of MDCK cells. Based on the previous test results that PR8-10 mAb was able to recognize HA on the membrane of the host cells in which the viruses were replicated, cell-based ELISA we established was good at analyzing the growth of virus in MDCK cells. Conclusion We obtained whole viral particles that were demonstrated to be able to stimulate the production of a high IgG titer in a mouse model with formalin-inactivated viral particles, and successfully prepared the mAb against H1N1 of high binding affinity and neutralization potency. HA-specific mAb can be used to analyze the characteristics of virus infection process and the effect of virus infection on the host cells as well.
目的 以纯化的病毒颗粒为抗原制备抗甲型流感病毒(H1N1)单克隆抗体(mAb),并利用该单克隆抗体研究流感病毒感染宿主细胞的特征。方法 A/PR/8(H1N1)病毒在鸡胚中培养,然后通过差速离心和密度梯度离心进一步纯化。通过透射电子显微镜(TEM)鉴定病毒颗粒的结构。用福尔马林灭活病毒免疫Balb/c小鼠,评估纯化病毒的免疫原性。通过Sp2/0骨髓瘤细胞与用该病毒免疫的小鼠脾细胞融合,建立分泌单克隆抗体的杂交瘤。通过酶联免疫吸附测定(ELISA)、免疫荧光测定(IFA)、蛋白质印迹法、血凝抑制试验(HI)和微量中和试验鉴定单克隆抗体的特性。使用流式细胞术检测病毒在其中增殖的宿主MDCK细胞膜上的外部血凝素(HA)以及感染病毒的MDCK细胞的凋亡情况。使用针对HA的单克隆抗体建立基于细胞的ELISA。随后,通过基于细胞的ELISA分析病毒的生长情况。结果 透射电子显微镜显示纯化病毒的物理结构为球形、椭圆形和细长丝状。小鼠血清中针对流感病毒的IgG滴度呈逐渐升高趋势,免疫6周后IgG滴度达到10。通过杂交瘤技术获得了6个分泌针对A/PR/8特异性单克隆抗体的杂交瘤克隆。PR8-10单克隆抗体的HI和中和活性显著高于其他单克隆抗体。PR8-10单克隆抗体的HI和中和滴度分别为1:2048和1:640。IFA和蛋白质印迹法证实PR8-10单克隆抗体可识别HA。流式细胞术显示PR8-10单克隆抗体也可识别病毒在其中复制的MDCK细胞膜上的HA,且病毒感染诱导MDCK细胞凋亡。基于之前PR8-10单克隆抗体能够识别病毒在其中复制的宿主细胞膜上的HA这一测试结果,我们建立的基于细胞的ELISA擅长分析病毒在MDCK细胞中的生长情况。结论 我们获得了经福尔马林灭活的病毒颗粒,其在小鼠模型中能够刺激产生高IgG滴度,并且成功制备了具有高结合亲和力和中和效力的抗H1N1单克隆抗体。HA特异性单克隆抗体可用于分析病毒感染过程的特征以及病毒感染对宿主细胞的影响。