Robust in vitro assay for analyzing the neutralization activity of serum specimens against hepatitis B virus.

ABSTRACT

Anti-HBs is a well-known marker of protective capability against HBV. However, little is known about the association between the qAnti-HBs determined by immunoassays and the neutralization activity (NAT) derived from functional assays. We developed an in vitro assay for direct measurement of the NAT of human sera. The new assay was highly sensitive, with an analytical sensitivity of 9.6 ± 1.3 mIU/mL for the HBIG standard. For serum detection, the maximum fold dilution required to produce ≥50% inhibition (MDF50) of HBV infection was used as the quantitative index. In vitro NAT evaluations were conducted for a cohort of 164 HBV-free healthy individuals. The results demonstrated that the NAT positively correlated with the qAnti-HBs (R2 = 0.473, p < 0.001). ROC analysis indicated that the optimal cutoff value of the qAnti-HBs to discriminate significant NAT (MDF50 ≥ 8) was 62.9 mIU/mL, with an AUROC of 0.920. Additionally, we found that the qAnti-HBc was another independent parameter positively associated with the NAT (R2 = 0.300, p < 0.001), which suggested that antibodies against other HBV proteins generated by previous HBV exposure possibly also contribute to the NAT. In summary, the new cell-based assay provides a robust tool to analyse the anti-HBV NAT. Abbreviations HBV Hepatitis B virus; HBsAg Hepatitis B surface antigen; Anti-HBs Hepatitis B surface antibody; HBeAg Hepatitis B e antigen; Anti-HBc Hepatitis B core antibody; qAnti-HBs quantitative hepatitis B surface antibody; qAnti-HBc quantitative hepatitis B core antibody; qHBeAg quantitative hepatitis B e antigen; NAT neutralization activity; HBIG hepatitis B immune globulin; NTCP Na+-taurocholate cotransporting polypeptide; IRES internal ribosome entry site; ccHBV cell culture derived hepatitis B virus; GE/cell genome equivalent per cell; MOI multiplicity of infection; Dpi day post infection; HepG2-TetOn a HepG2-derived cell line that expresses the doxycycline-regulated transactivator; ROC receiver operating characteristic curve; AUROC area under receiver operating characteristic curve; LLOQ the lower limits of quantification; MDF50 the maximum fold dilution required to produce ≥50% inhibition; IC50 half maximal inhibitory concentration.