| Chickenpox is caused by varicella-zoster virus (VZV), and mainly seen in children. The disease is highly contagious, and sometimes accompanied by variety of complications. After chickenpox infection, the re-activation of latent virus in the sensory nerve center can cause shingles. At present, there is no effective treatment against chickenpox and shingles. However, the vaccine can be effective in preventing chicken pox and shingles. As the application of varicella vaccine increased, the high sensitivity, specificity, fast and simple methods were needed to evaluate the comprehensive immune effect of the vaccine. The methods eatablished to detect VZV special antibody had many disadvantages and most of them were qualitative or semi-quantitative. The cell-mediated immunity which was considered playing a key role in clearing VZV and preventing shingles had not been applied in assessing the immune responses to varicella vaccine. In this study, glycoprotein ELISA (gp-ELISA) method was established to detect VZV special IgG antibody in serum using purified VZV glycoprotein as coating antigen, and ELISPOT method was established to detect the effectiveness of cell-mediated immunity in mice. Immune effect of live attenuated varicella vaccine and inactivated vaccine was compared by these two methods.VZV glycoprotein was purifed from VZV infected cells by Lentil Lectin Sepharose 4B, and then was used as a coating antigen to establish gp-ELISA method. The gp-ELISA was established by optimizing the reagent buffer and procedures for detection, such as the coated concentration of antigens, the blocking buffer ingredients, the diluting buffer of serum sample and secondary antibody, the reaction time of serum sample and secondary antibody, the concentration of secondary antibody and the reaction time of substrate. For quantitative detection purpose, a diluted VZV IgG reference was used as the standard to calibrate the unknown samples, and the standard curve regression equation was y=1.2878x+0.1481, r2 resched to 0.9939, the detection limit was 1.86 mIU/mL, while the cutoff value was 6.13 mIU/mL. At the same time,153 children's sera were detected using classical FAMA, Serion's VZV IgG diagnostic kit and gp-ELISA method. Compared to FAMA, the concordance rate of gp-ELISA was 95.8% for negative sera,77.6% for positive sera. These data showed that the established gp-ELISA had satisfying specificity, stability and precision.Mice were immunized with both live attenuated varicella vaccine and components of MRC-5 cells prepared in the same way at the same time. After two weeks, mouse spleen lymphocytes were separated, and the cell responsive frequency to IL-2 and IFN-y were detected with ELISPOT. The cell concentration was 1×106/pool, the dose of vaccine was 3750PFU and the stimulus concentration was 100μg/mL of VZV-gp,50μg/mL of Cell-gp.The mice were immunized with live attenuated varicella vaccine and inactivated vaccine, respectively.2,3and 4 weeks later, the cell responsive frequency to IL-2 and IFN-y were detected with ELISPOT, and VZV special IgG antibody in serum were detected with gp-ELISA. The results showed that there was cell-mediated immunity response to both vaccine within four weeks and had no significant differences, there were also no significant differences for VZV specific IgG antibody level within three weeks. However, in the fourth week, antibody levels induced by live attenuated vaccine were significantly higher than that induced by inactivated vaccine.In this study, a gp-ELISA method was successfully established to quantitatively detecte VZV specific IgG antibody in serum. This method was a high sensitivity, good specificity, good stability and precision method. The established ELISPOT method could detect cell-mediated immunity to varicella vaccine, and it is also high sensitivity, good specificity, good stability and precision. The establishment of these two methods provided a basis for a comprehensive evaluation of the effect of varicella vaccine. |
PDF Full Text Request