ABSTRACT
Objective:To prepare and screen monoclonal antibodies against Herpes simplex virus-1(HSV-1),and develop a double antibody sandwich quantitative enzyme-linked immunosorbent assay( Q-ELISA) for detection of HSV-1 particle. This method was used to control the quality of viral particle in the developing and manufacturing process of HSV-1. Methods: BALB/c mice was immunized with HSV-1 to prepare monoclonal antibodies. A double antibody sandwich Q-ELISA was developed to determine concentration of HSV-1 particle,which was based on the neutralizing monoclonal antibody 1F6 as capture antibody,and 2B1 as HRP-conjugated antibody. The performance of the reagent was evaluated,including specificity,sensitivity,precision,accuracy and linear. And the relation between the amount of virus detected by this method and the virus titer was analyzed by regression analysis method. Results: The Q-ELISA for HSV-1 particle was developed. The quantitation scope was 0. 125-2 μg/ml, the coefficient correlation was 0. 995 5, the limit of detection was 0. 125 μg/ml, the recovery was between 85. 6% and 107. 1%, the variation coefficient was lower than 10%, and the reagent does not react with other samples except HSV-1 antigen. This method has a good correlation with virus titer. Conclusion:The Q-ELISA for HSV-1 particle was successfully developed,which provide a new approach for rapid and quantitative detection of HSV-1 antigen.
ABSTRACT
ObjectiveTo develop an ELISA method for quantitative determination of enterovirus 71 (EV71) antigen.The method can be applied to detect EV71 antigen contents and analyze the correlation between immunogenicity and immunoprotection.It also can be used for tissue culture infective dose( TCID50 ) assay.MethodsA double antibody sandwich ELISA method was developed for quantitative determination of EV71 antigen on the basis of the high-affinity neutralizing monoclonal antibodies ( K8G2 and Y8H2-HRP).This method was compared with microscopic observation for the detection of EV71 TCID50.The correlation was analyzed between the specific activity of EV71 antigen and the EV71 neutralizing antibody titer in immune serum.ResultsThe linear range of this method was 0.125-4.0 U/ml and the R2 value was 0.9911.The reagent did not react with other antigens except EV71 antigen.The recovery ratio of this method was 0.89-1.16.The coefficient of variation was less than 15％.The heat recovery rate was above 85％ when the reagent was in 37℃ for 9 days.There was a good correlation in TCID50 of EV71 between this method and microscopic observation,r=0.990.The specific activity of EV71 antigen had positive correlation with the neutralization titer of immune serum in 21 EV71 strains,r=0.930.ConclusionThe quantitative ELISA method for EV71 antigen was developed,which could be used to detect EV71 antigen contents and analyze TCID50.The specific activity of EV71 antigen detected by the method could be used to evaluate the immunoprotection of the vaccine potency test.
ABSTRACT
Objective To screen the 5 EV71 vaccine candidates which were isolated from MRC-5 cells to find one as the vaccine virus. Methods The ICR mother mouse were immunized by intraperitoneal injection with the 5 vaccine candidates which were made from monoclonal EV71 virus. Two weeks after booster immunization, the animals were allowed to mate, another booster was given after 2 weeks, and then attracted the milk mouse within 24 h with different types of virus by cranial cavity injection. The survival condition were recorded everyday, and the antibody titre(IgG) were detected by ELISA, the virus titre of intestine were detected by nest-PCR, and neutralizing antibodies were determined using a microassay with MRC-5 cells, and then the data were analyzed by SPSS16.0. Results The antibody titre of 5 virus immunized ICR mouse were improved with the increase in the immune times, and they got difference in neutralization capacity, the survival rate after fatal attract and the virus titre of the intestine. Conclusion It proved that the five vaccine candidates were different at the molecular level, cellular level and individual level. 123 strain was the best one in immunogenicity and immunoprotective property, which agreed with the vaccine requirement.