[Preparation and identification of monoclonal antibody against H1N1 influenza virus]

Abstract

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 10⁶ 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.