Adaptation And Passages Of Varicella-Zoster Virus Vaccine Strain In Vero Cell Cultures And Establishment Of ELISA To Detect IgG In Serum

Varicella is a epidemic disease worldwide caused by infection of Varicella-Zoster virus (VZV). In present thesis, adaptation and passages of VZV vaccine strains in Vero cell cultures, antigen preparation and ELISA were studied.The VZV was inoculated and continuously passaged in Vero cells. The adaptation of virus to Vero cells was observed from the 9th passage expressed in the cytopathic effect (CPE) more significant and the titers of virus increased from 2~3 log PFU/ml to≧4.2 log PFU/ml. The virus vaccine strain was stable under storage at–70℃, and could be used to prepare VZV antigen. The optimal propagation conditions of VZV were tested. In 15L roller bottle, 0.01 of MOI (multiplication of infection, virus/cells, PFU/cell) was inoculated, the infected cells were harvested at 60~90 hrs after inoculation. The harvested cells were ultrasonically crashed and centrifuged, and Total Virus ELISA (VAR ELISA) antigen was yield. The cells was treated with Triton X-100, following affinity chromatography with Lentil Lectin Sephrose 4B gel, VZV glycoprotein ELISA (gpELISA) antigen was purified, and showed specific VZV glycoprotein.The conditions for indirect VAR ELISA and gpELISA tests were studied, and ELISA methodology was established. The antigen with optimal working dilution ratio of 1:4000 was coated with PBS buffer (pH7.4) and blocked with 10% NCS, and the reacted with antibody for 120min. The enzyme labeled antibody was added with optimal dilution of 1:120, and bounded with horseradish peroxidase (HRP) for 120 min. O-phenylenediamine was used the substrate of HRP. VZV VAR and gpELISA antigens with stabilizer kept stable during 8 months at -20℃. When the WHO International Reference Preparations anti-VZV standard was detected, gpELISA was sensitive four times than VAR ELISA. The lowest limits of detection were 195mIU/ml with VAR ELISA and 781mIU/ml with gpELISA, respectively. The results were consistent with those obtained from commercial imported ELISA Kits. The anti-VZV positive serum was obtained from screening 210 samples of blood donor. The anti-VZV IgG GMT of Vero gpELISA and VAR ELISA were superior to that of 2BS VAR ELISA.Adaptation and passage of vaccine strain of VZV to Vero cells allowed to culture in 15L roller bottles, and VZV antigen could be successfully prepared. VZV gpELISA and VAR ELISA were stable, specific and sensitive with convenient usage. It is practicable to research VZV and prepare diagnostic kit using Vero cells. This study allowed the bases on standardization and commercial application of Vero gpELISA and VAR ELISA kits.