Enzyme linked immunosorbent assay kit

From September 2006 to August 2012, 285,858 residents undergoing a physical examination at the physical examination department of the local centers for disease prevention and control were enrolled into this study from major cities in Northwest China, i.e., Lanzhou City of Gansu Province, Xi’an City of Shaanxi Province, Xining City of Qinghai Province, Urumuqi City of the Xinjiang Autonomous Region and Yinchuan City of the Ningxia Autonomous Region. HBV serologic markers were tested using commercially available enzyme-linked immunosorbent assay kits (Abbott Laboratories, USA; Beijing Wantai Co., Ltd, Beijing). Information about HB-related medical history was collected by trained investigators. The serum viral load was measured with qualitative assays (Roche Ltd., Switzerland). Only patients positive for HBsAg, having no HB-related medical history or typical clinical symptoms, and with HBV DNA ≥ 10⁵ IU/ml were enrolled. Approval was obtained from the local and Fourth Military Medical University institutional ethics committee before the study, and informed consent was obtained from each individual. All serum samples were collected and stored at −80°C before use.

After obtaining consent, we collected a 5 ml peripheral blood specimen from each patient in ethylene diamine tetraacetic acid coated tubes (BD Vacutainers, Franklin Lakes, NJ, USA). The plasma was isolated within 24 hours for determination of the HBsAg by commercial enzyme-linked immunosorbent assay kits (Abbott Park, Wiesbaden, Germany) and the rest of blood specimen was used for extraction of genomic DNA by commercial kit (Axygen Scientific Inc, Union City, CA, USA). The DNA samples were stored at −80 °C until further analysis.

HBsAg, hepatitis B e antigen, and antibody to hepatitis B e antigen were tested by use of commercially available enzyme-linked immunosorbent assay kits (Abbott Laboratories, North Chicago, IL, USA). Anti-HCV was determined by a third-generation enzyme immunoassay (Abbott Laboratories). HCV RNA was measured by use of a qualitative polymerase chain reaction assay, with a detection limit of 50 IU/mL (CobasAmplicor Hepatitis C Virus Test, Version 2.0; Roche Diagnostics, Branchburg, NJ). Serum levels of HCV RNA were quantified by the branched DNA assay, with a quantification limit of 615 IU/mL (Versant HCV RNA 3.0, Bayer, Tarrytown, NJ) if qualitative HCV RNA was seropositive. HCV genotypes were determined by use of the method described by Okamoto et al. [24 (link)]. Serum HBV DNA levels were determined by use of the CobasAmpliPrep/CobasTaqMan HBV assay, with a dynamic range of 20 IU/mL–1.7 × 10⁸IU/mL (CAP/CTM Version 2.0, Roche Diagnostics, Indianapolis, IN, USA). Stored serum was used for the HBV DNA test, if available. Liver cirrhosis was diagnosed by either histology or ultrasound diagnosis combined with evidence of portal hypertension, such as splenomegaly, esophageal, or gastric varices.

Hepatitis B surface antigen (HBsAg) was identified and determined through standard quantitative chemiluminescent micro-particle immunoassay (ARCHITECT HBsAg, Abbott Diagnostics) or qualitative assay (Abbott Laboratories, North Chicago, IL, USA). Hepatitis B e-antigen (HBeAg) was identified and determined using enzyme-linked immunosorbent assay kits (Abbott Laboratories). HBV DNA from the serum was determined using a standardized, automated quantitative PCR assay (COBAS TaqMan HBV test, Roche Diagnostics, Branchburg, NJ; detection limit 12 IU/mL) [33 (link)]. Anti-HDV immunoglobulin G (IgG), examined by using an anti-HDV enzyme-linked immunosorbent assay kit (General Biologicals Corporation, Taiwan) [4 (link)], was checked prior to initiating NUCs therapy, and patient serology with anti-HDV seropositivity was monitored annually from there on. HDV RNA was examined in patients seropositive for anti-HDV using a LightMix Kit HDV (Berlin, Germany) on a Roche LightCycler (detecting limit: 10 copies per mL) [18 (link)].