Robust <i>in vitro</i> assay for analyzing the neutralization activity of serum specimens against hepatitis B virus
Creators: name: Zhang, Ya-Li
name: Gao, Ying
name: Cao, Jia-Li
name: Zhao, Jing-Hua
name: Zhang, Tian-Ying
name: Yang, Chuan-Lai
name: Xiong, Hua-Long
name: Wang, Ying-Bin
name: Ou, Shan-Hai
name: Cheng, Tong
name: Chen, Chang-Rong
name: Yuan, Quan
name: Xia, Ning-Shao
Publisher: name: Taylor & Francis
Year: 2019
Subjects: subject: Biophysics
subject: Biochemistry
subject: Cell Biology
subject: Molecular Biology
subject: Biotechnology
subject: Sociology
subject: FOS: Sociology
subject: Chemical Sciences not elsewhere classified
subject: Immunology
subject: FOS: Clinical medicine
subject: Cancer
subject: Hematology
subject: Infectious Diseases
subject: FOS: Health sciences
subject: Virology

description: 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 <i>in vitro</i> 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 (MDF<sub>50</sub>) of HBV infection was used as the quantitative index. <i>In vitro</i> 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 (<i>R</i><sup>2</sup> = 0.473, <i>p</i> &lt; 0.001). ROC analysis indicated that the optimal cutoff value of the qAnti-HBs to discriminate significant NAT (MDF<sub>50</sub> ≥ 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 (<i>R</i><sup>2</sup> = 0.300, <i>p</i> &lt; 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. <b>Abbreviations:</b> 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<sup>+</sup>-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; MDF<sub>50</sub>: the maximum fold dilution required to produce ≥50% inhibition; IC50: half maximal inhibitory concentration