HBV serological markers

HBsAg concentrations were quantified using the Abbott ARCHITECT i4000SR chemiluminescent microparticle immunoassay (Abbott Diagnostics, Abbott Park, IL, USA), which has a detection range of 0.05 to 250 IU/mL. The samples with concentrations >250 IU/mL were used at a dilution of 1: 500 and retested.

Hepatitis B e antigen (HBeAg) concentrations were tested using commercially available enzyme immunoassay kits (Abbott Diagnostics). HBV-DNA in serum samples was detected and quantified using the Amplly real-time polymerase chain reaction HBV assay performed on an Anadas9850 platform (Amplly Engineering Co. Ltd., Xiamen, China), which is a fully-automated nucleic acid extraction and real-time polymerase chain reaction system. Using the Amplly assay, HBV DNA was extracted from 200 μL of serum and amplified using the Anadas9850. The limit of detection was approximately 20 IU/mL. The samples with <20 IU/mL were regarded as undetectable in accordance with the manufacturer’s instructions.

Surplus serum samples from patients were stored at −20°C and assayed using a fully automatic chemiluminescent enzyme immunoassay system (Lumipulse G1200; Fujirebio, Tokyo, Japan) according to the manufacturer’s instructions. Briefly, the assay provided a linear range of 3 to 7 log₁₀ U/mL. However, the system indicates concentrations <3 log₁₀ U/mL down to 2 log₁₀ U/mL in HBcrAg-positive samples. HBcrAg concentrations <2 log₁₀ U/mL were treated as 2 log₁₀ U/mL for statistical analysis. Samples with HBcrAg concentrations >7 log₁₀ U/mL were used at a dilution of 1:100 or 1:1000, according to the results of HBeAg, with the sera of healthy controls and retested to quantify HBcrAg concentrations.