| Hepatitis B virus infection is an important public health problem in China and around the world.About 2 billion people worldwide have been infected with HBV,and more than 400 million people are chronic hepatitis B virus carriers.Each year about 650,000 people died of liver cirrhosis,liver cancer and other HBV infection related diseases.However,the current antiviral therapy can only induce disease remission,but hardly eradicate HBV effectively.Therefore,vaccination of preventive hepatitis vaccine is essential to prevent HBV infection,reduce the population HBsAg carrying rate.Individuals after injection of vaccines produce different levels of antibodies,and different antibody neutralizing activities determine the immunization of host against HBV.About 10-15%of the population after vaccination are without response or low response,in this case need revaccination in order to produce protective antibodies,thus resist HBV invasion and prevent HBV infection.Currently,common methods of evaluating neutralizing antibody activity is mainly based on "binding activity" immunoassay,such as ELISA,however that cannot truly reflect the neutralization activity of the sample.In this study,a more accurate and reliable neutralizing antibody titer detection assay was constructed based on HepG2-NTCP which is a hepatitis B virus infected cell model,so as to achieve a large sample size antibody neutralization activity evaluation.HepG2-NTCP,the hepatocarcinoma cells overexpression of NTCP,can be used as a cell model of hepatitis B virus infection to achieve HBV infection in vitro.The cell model has the advantages of simple operation,good reproducibility,short culture period,and large scale to cultivate.In this study,the neutralization activity assay was based on HepG2-NTCP cell model,and HBIG was used as a positive control to test the best infection dose of 1.25 GE/cell and the post-infection supernatant detected time was 9 days post infection.The residual virus infectivity of the sample was obtained by detecting the HBeAg level in the supernatant,and the neutralization antibody titer(NAT)of the sample to be tested was the dilution factor closest to the 50%inhibition of the control group.Three experiments were performed with a CV of 10%,indicating that the assay was reproducible.In this study,the detection of neutralizing antibody titers for serum samples was mainly dependent on the dilution method,which was more suitable for the detection of larger-scale samples than IC50 calculation.In present study,the neutralization activity assay were used to evaluate the neutralized antibody titers of 164 healthy blood donors from Xiamen blood bank.The neutralization antibody titers were ranging from 1 to 256.And detected the Anti-HBs levels and Anti-HBc levels of the 164 volunteers’ serum samples.The results showed that the level of Anti-HBs was closely related to the neutralizing antibody titer(Pearson-0.70),moreover Anti-HBs from different origins had a good protective effect on HBV infection,which was statistically significant.However,in the healthy individuals with no significant differences in Anti-HBs(p = 0.57),serum neutralizing activity of the previously infected population(ie,Anti-HBc positive population)(log10(NAT)mean 1.05)was superior to those never been infected with HBV(log10(NAT)mean of 0.77),the difference was statistically significant.Therefore both consider Anti-HBs,Anti-HBc levels can better assess the level of neutralizing antibody titer.In addition,the Anti-HBs level was detected by two kinds of Anti-HBs detection methods.It was found that the quantitative results of double antigen sandwich enzyme-linked immunosorbent assay and chemiluminescence microfilm immunization were basically the same(R = 0.91,Kappa value of 0.84),both are good to reflect the level of Anti-HBs.In summary,we used the HepG2-NTCP cell model to develop a neutralizing activity assay to detect the neutralizing antibody titer(NAT)of the samples.The assay can achieve a large sample scale of the antibody neutralization activity assay,can be used by detection of serum neutralizing antibody titers after vaccination,and also by monoclonal/polyclonal antibody neutralization activity evaluation to screen new antiviral drugs and so on.In addition,in the healthy population,we can both consider Anti-HBs,Anti-HBc levels to initially evaluate the vaccine against HBV protection in the absence of detection of serum HBV neutralizing antibody levels. |
PDF Full Text Request