Elecsys hbsag 2 quant assay

25(OH)D₃ levels in serum samples were measured using an automated electrochemiluminescence-based assay, Elecsys Vitamin D Total (Roche Diagnostics, Mannheim, Germany). The data were expressed in nanograms per milliliter. Serum 25(OH)D₃ concentrations of <10 ng/mL, <20 ng/mL and ≥20 ng/mL were defined as vitamin D deficiency, insufficiency and sufficiency, respectively.
The serum calcium was measured with the inductively coupled plasma mass spectrometry, while the serum phosphorus concentration was measured by phosphomolybdic acid colorimetry method. Serum alanine aminotransferase (ALT) and alkaline phosphatase (ALP) were measured using the Automatic Biochemistry analyzer (Olympus AU5400, Olympus Corporation, Tokyo, Japan). Serum intact parathyroid hormone (iPTH) was measured by a two-site immunoradiometric assay. Serum HBV DNA was measured using the Cobas Taqman assay (Roche Diagnostics, Branchburg, NJ) with a lower limit of detection of 291 copies/mL. HBV genotype was measured by directly gene sequencing of HBV DNA S gene. Serum HBsAg titres were quantified according to research protocol using Elecsys® HBsAg II Quant Assay (Roche Diagnostics, Penzberg, Germany). Serum HBeAg was measured using commercially available immunoassays (Roche Diagnostics, Indianapolis, IN, USA).

Serum ALT levels were assessed according to standard procedures (Olympus AU5400, Olympus Corporation, Tokyo, Japan), and the upper limit of normal (ULN) ALT was defined as 50 IU/L for men and 38 IU/L for women. Serum HBsAg concentration was quantitatively assessed using Elecsys® HBsAg II Quant Assay (Roche Diagnostics, Penzberg, Germany), with a dynamic range of 20 to 52,000  IU/mL. If qHBsAg levels >52,000  IU/mL, samples were retested with a stepwise dilution of 1:4000. Serum hepatitis B e antigen (HBeAg) was determined by the electrochemiluminescence immunoassay (Roche Diagnostics, Indianapolis, IN, USA). Serum concentrations of HBV-DNA were determined using Cobas Taqman assay kit (Roche Diagnostics, Branchburg, NJ), with a lower limit of detection of 20 IU/mL. HBV genotypes were determined by direct S-gene sequencing.

Blood collections were performed at study entry and follow-ups. Tests on serum markers were performed using commercial kits: HBsAg and HBeAg by radioimmunoassay (Abbott Laboratories, North Chicago, IL), ALT by serum chemistry autoanalyzer (model 736; Hitachi Co., Tokyo, Japan) using commercial reagents, serum HBV DNA levels by polymerase chain reaction (COBAS Amplicor; Roche Diagnostics, Indianapolis, IN) for baseline samples, and by real-time polymerase chain reaction (COBAS TaqMan; Roche Diagnostics, Indianapolis, IN) for follow-up samples. Serum HBsAg levels were quantified using the Elecsys HBsAg II Quant assay (Roche Diagnostics GmbH, Mannheim, Germany). M2BPGi was quantified with a novel sandwich immunoassay using the fully automated chemiluminescence enzyme immunoanalyzer, HISCL-5000 (Sysmex Co., Kobe, Japan)^{21 (link)}. Raw counts of M2BPGi were converted to a standardized cut-off index (COI) for analysis^{32 (link)}.