Serum samples collected were stored at –20°C until tested. Baseline was defined as the time of flares for severe AFOCHB group and as the time of matching for control group. qHBsAg levels were measured at baseline and subsequently on a yearly basis. qHBsAg levels were measured using the Elecsys HBsAg II assay (Roche Diagnostic, GmbH, Mannheim, Germany),17 (link) with lower limit of quantification of 0.05 IU/mL. Samples above 52,000 IU/mL were retested at a dilution of 1:100, according to manufacturer’s instructions. For patients with undetectable qHBsAg, the values taken for statistical analyses were 0.05 IU/mL. Serum HBsAg seroclearance was defined as undetectable qHBsAg for two samples taken at least 6 months apart. HBeAg seroconversion was defined by the loss of HBeAg and development of antibody to HBeAg. Qualitative HBeAg was measured by Abbott Laboratories (Chicago, IL, USA). HBV DNA levels were measured using the Cobas Taqman assay (Roche Diagnostics, Branchburg, NJ, USA), with the lower limit of detection of 10 IU/mL.
Cobas taqman assay
Manufactured by Roche
Sourced in United States, Switzerland, Germany
The COBAS TaqMan assay is a laboratory equipment product designed for nucleic acid detection and quantification. It utilizes real-time PCR technology to accurately measure the presence and amount of specific genetic targets in samples. The core function of the COBAS TaqMan assay is to provide reliable and precise diagnostic results for various clinical and research applications.
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Lab products found in correlation
Elecsys hbsag 2 quant assay, by Roche (3 mentions)
Architect i2000 assay, by Abbott (3 mentions)
Architect hbsag, by Abbott (2 mentions)
Cobas amplicor monitor 2.0 assay, by Roche (2 mentions)
Nasba nuclisens, by Organon (2 mentions)
Elecsys hbsag 2, by Roche (1 mentions)
Elecsys platform, by Roche (1 mentions)
Architect immunoassay, by Abbott (1 mentions)
Line probe assay, by Fujirebio (1 mentions)
Elecsys hbsag 2 assay, by Roche (1 mentions)
Axsym, by Abbott (1 mentions)
Bigdye terminator version 3.1 cycle sequencing kit, by Thermo Fisher Scientific (1 mentions)
Enzyme linked immunosorbent assay, by Abbott (1 mentions)
Abi 3730 sequencer, by Thermo Fisher Scientific (1 mentions)
Lightcycler 480, by Roche (1 mentions)
Architect i2000, by Abbott (1 mentions)
Hbsag eia, by Abbott (1 mentions)
Hbeag eia, by Abbott (1 mentions)
Cobas ampliprep cobas taqman hcv kit, by Roche (1 mentions)
Architect i2000sr system, by Abbott (1 mentions)
36 protocols using cobas taqman assay
1
Quantitative HBsAg Monitoring in Chronic Hepatitis B
2
Comprehensive HBV-HIV Coinfection Evaluation
3
Quantifying HBsAg and HBV DNA Levels
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HBsAg was quantified by using a standard quantitative chemiluminescent microparticle immunoassay (Architect HBsAg, Abbott Diagnostics, Princeton, NJ, USA). The concentration of HBsAg in the specimen was determined using a previously generated Architect HBsAg calibration curve (range, 0.05–250 IU/mL). Serum HBsAg < 0.05 IU/mL was defined as clearance of HBsAg. Samples with serum HBsAg titer >250 IU/mL were diluted to 1:20 and 1:500 with the Architect HBsAg diluent and retested to expand the upper limit of the dynamic range from 250 to 125,000 IU/mL. HBV DNA levels were quantified using the Cobas Taqman assay (Roche Diagnostics, Basel, Switzerland), which has a lower limit of quantification of 60 copies/mL (12 IU/mL) and a linear range of upper detection limit of 6.4 × 108 copies/mL (1.3 × 108 IU/mL). For results exceeding the upper detection limit, HBV DNA was remeasured after 100,000-fold dilution.
4
Comprehensive HBV Resistance Profiling
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Serum HBV DNA levels were measured using Cobas Taqman assay (Roche Diagnostics, Branchburg, NJ) with the lower limit of detection of 20 IU/ml. Resistance profile was performed using a line probe assay (Innogenetics NV, Gent, Belgium), with both line probe assay DR version 2 and 3 used to identify the amino acids at codons rt173, rt180, rt240 and rt184, and rt202 and rt250, respectively. Genotypic resistance to entecavir was defined by the presence of three viral mutations: rtL180M, rtM204V/I, and one of the following: rtT184S/C/G/A, rtS202G/C/I, or rtM250V. Serum HBeAg, antibody to HBeAg and antibody to HBsAg were measured by the Architect Immunoassays (Abbott Laboratories, Chicago, IL). Serum HBsAg levels were performed using Elecsys HBsAg II Assay (Roche Diagnostics, Branchburg, NJ), with a linear range of 0.05–52,000 IU/ml. Samples with levels higher than 52,000 IU/ml were tested at a dilution of 1:100 according to the manufacturer’s instruction. The HBcrAg levels were determined by the Lumipulse G HBcrAg Chemiluminescence Enzyme Immunoassay (Fujirebio, Tokyo, Japan). The dynamic range of the assay ranges from 1 to 1 × 104 kU/ml. Samples with HBcrAg>104 kU/ml were retested at dilution of 1:100 according to the manufacturer’s instruction. In the present study, the lower cutoff value of HBcrAg concentration is 1 kU/ml.
5
Comprehensive Hepatitis B Laboratory Analysis
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Biochemical, hematological and virological laboratory analysis were carried out by the local diagnostic laboratory in accordance with good laboratory practice. ALT levels were expressed as absolute values (U/L) or relative to the ULN range (45 U/L for men and 34 U/L for women). HBV-DNA levels in plasma were determined using the COBAS TaqMan assay (F Hoffmann-La Roche, Basel, Switzerland). Qualitative detection of serum HBsAg, antibody to HBsAg (anti-HBs), HBeAg, and antibody to HBeAg (anti-HBe) was done using an enzyme immunoassay (AxSYM; Abbott Laboratories, Abbott Park, IL, USA). Quantified serum HBsAg measurement was carried out by Sanquin Diagnostics (Amsterdam, the Netherlands) using the Abbott HBsAg Architect assay (Abbott Diagnostics, Abbott Park, IL, USA). The modified Ishak scoring system was used for histological assessment of liver biopsies in the initial study.
6
Long-term Follow-up of Entecavir Therapy
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Liver biochemistry, HBV serological markers, serum HBV DNA, and HBsAg titers were measured before (baseline) and after 3 months, 6 months, 1 year, 2 years, 3 years, 4 years, 5 years, 6 years, and 7 years of ETV therapy.
After overnight (12 h) fasting, venous blood was drawn to determine the serum levels of alanine aminotransferase (ALT) using an automatic biochemical analyzer. Serology markers for HBV, antibody to HBsAg; hepatitis B e antigen (HBeAg); and antibody to HBeAg were measured by enzyme-linked immunosorbent assay (Abbott Laboratories, Chicago, IL, USA).
Serum HBsAg titers were measured using the Elecsys HBsAgII quant assay (Roche Diagnostics, Branchburg, NJ, USA) with a detection limit of 0.05–52,000 IU/ml. Serum HBV DNA levels were determined using the COBAS® TaqMan assay (Roche Diagnostics) with a detection limit of 20 IU/ml.
The HBV genotype was determined by direct sequencing of the preS/S gene. HBV DNA was amplified as previously described.[17 (link)] Polymerase chain reaction products were sequenced in both directions using the Big-Dye Terminator version 3.1 Cycle Sequencing kit on the ABI 3730 sequencer (Applied Biosystems, Foster City, CA, USA). HBV genotypes were determined by comparing the generated preS/S gene sequences with prototype sequences from the GenBank using a web-based genotyping tool (National Center for Biotechnology Information).
After overnight (12 h) fasting, venous blood was drawn to determine the serum levels of alanine aminotransferase (ALT) using an automatic biochemical analyzer. Serology markers for HBV, antibody to HBsAg; hepatitis B e antigen (HBeAg); and antibody to HBeAg were measured by enzyme-linked immunosorbent assay (Abbott Laboratories, Chicago, IL, USA).
Serum HBsAg titers were measured using the Elecsys HBsAgII quant assay (Roche Diagnostics, Branchburg, NJ, USA) with a detection limit of 0.05–52,000 IU/ml. Serum HBV DNA levels were determined using the COBAS® TaqMan assay (Roche Diagnostics) with a detection limit of 20 IU/ml.
The HBV genotype was determined by direct sequencing of the preS/S gene. HBV DNA was amplified as previously described.[17 (link)] Polymerase chain reaction products were sequenced in both directions using the Big-Dye Terminator version 3.1 Cycle Sequencing kit on the ABI 3730 sequencer (Applied Biosystems, Foster City, CA, USA). HBV genotypes were determined by comparing the generated preS/S gene sequences with prototype sequences from the GenBank using a web-based genotyping tool (National Center for Biotechnology Information).
7
Quantification of Hepatitis B Biomarkers
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An automatic biochemical analyzer was used to test the biochemical indicators, including alanine aminotransferase (ALT) and aspartate transaminase (AST). Quantitative HBsAg, HBeAg, and anti-HBe were determined with ELISA kits (Abbott Laboratories, Chicago, IL, United States). COBAS TaqMan assay (Roche Diagnostics, Basel, Switzerland) was used to quantify serum HBV DNA levels, with 100 IU/mL as the lower limit of detection. Identification of HBV genotypes was performed by comparison with the generated preS/S gene sequences in GenBank (NCBI) data[9 (link)].
8
Evaluating Antiviral Therapy Outcomes in Liver Transplant Recipients
9
Comparative Analysis of HBV DNA Quantification
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The Daan test was performed at the Nanfang Hospital per the manufacturer’s instructions (Daan Gene Co, Ltd of Sun Yat-sen University, Guangdong, China). Briefly, HBV DNA was extracted with control DNA from 100 μL of serum and detected by PCR fluorescence probing. The real-time PCR assay was performed in the Roche LightCycler 480 (Roche Diagnostics Ltd,Switzerland) according to the manufacturer’s instructions; the PCR amplification reaction used thermus aquaticus DNA polymerase, deoxyribonucleoside triphosphates (dNTPs), and a HBV DNA specific probe to detect and measure serum HBV DNA levels. The manufacturer reports a linear range of 1 × 102 IU/mL to 1 × 108 IU/mL.
The Cobas TaqMan assay was performed at the Nanfang Hospital according to the manufacturer’s instructions (Roche Molecular Systems, Inc, Pleasanton, CA, USA). The Cobas TaqMan assay is based on the co-amplification of target HBV DNA and the detection of a cleaved dual-labeled oligonucleotide detection probe specific to the target. Serum samples of 750 μL were transferred for automatic processing. The manufacturer reports a HBV DNA linear range of 20 IU/mL to 1.7 × 108 IU/mL.
The HBV genotype was determined by nested PCR with the Qiagen Mini Kit (Qiagen), following the manufacturer’s instructions.
The Cobas TaqMan assay was performed at the Nanfang Hospital according to the manufacturer’s instructions (Roche Molecular Systems, Inc, Pleasanton, CA, USA). The Cobas TaqMan assay is based on the co-amplification of target HBV DNA and the detection of a cleaved dual-labeled oligonucleotide detection probe specific to the target. Serum samples of 750 μL were transferred for automatic processing. The manufacturer reports a HBV DNA linear range of 20 IU/mL to 1.7 × 108 IU/mL.
The HBV genotype was determined by nested PCR with the Qiagen Mini Kit (Qiagen), following the manufacturer’s instructions.
10
Comprehensive Hepatitis B Serological Assessment
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HBsAg qualitative and quantitative, anti-HBs, anti-HBc, IgM anti-HBc HBeAg and anti-HBe, anti-HCV, anti-HDV and anti-HIV were detected by commercially available immunoassays (Abbott laboratories, N. Chicago, IL, USA). Serum HBV DNA levels were quantified by COBAS TaqMan assay, sensitivity 6 IU/mL, dynamic range 6–1.70×108 IU/mL (Roche Diagnostic Systems Inc, Mannheim, Germany). HBV genotyping was performed by direct sequencing of the small HBs region. Serum ALT levels were tested by routine biochemistry (normal range: <45 U/L and <33 U/L for male and female respectively). Circulating HBsAg particles were obtained by immunoprecipitation as previously reported [14] (link).
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