Alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were measured using an Olympus Automatic Biochemical Analyzer AU5400 (Olympus, Tokyo, Japan) with a cut-off value of 40 IU/L. The viral markers, including serum HBsAg, HBeAg and anti-HBe, were determined using an electrochemiluminescence immunoassay (Abbott Laboratories, Chicago, IL, USA). The serum HBV DNA level was determined using the Cobas HBV Amplicor Monitor assay (Roche Diagnostics, Pleasanton, CA, USA), with a lower limit of detection of 2.46 log10 copies/mL (~50 IU/mL or 291 copies/mL).
Cobas hbv amplicor monitor assay
Manufactured by Roche
Sourced in United States
The Cobas HBV Amplicor Monitor assay is a laboratory diagnostic tool used for the quantitative detection of hepatitis B virus (HBV) DNA in human blood samples. It employs polymerase chain reaction (PCR) technology to amplify and quantify HBV genetic material.
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Lab products found in correlation
Au5400 automatic biochemical analyzer, by Olympus (3 mentions)
Architect i2000 system, by Abbott (2 mentions)
Dnase 1, by Thermo Fisher Scientific (2 mentions)
Electrochemiluminescence immunoassay, by Abbott (1 mentions)
Nucleic acid extraction or purification kit, by Sansure Biotech (1 mentions)
Elecsys hbsag 2 quant reagent kits, by Roche (1 mentions)
Pyromark q24 mdx system, by Qiagen (1 mentions)
Axsym system, by Abbott (1 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (1 mentions)
9 protocols using cobas hbv amplicor monitor assay
1
Hepatitis B Biomarker Evaluation
2
Hepatitis B Viral Marker Analysis
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Alanine aminotransferase and aspartate aminotransferase levels were measured using an Olympus Automatic Biochemical Analyzer AU5400 (Olympus Corp., Tokyo, Japan) with a cut-off value of 40 IU/l, according to the manufacturer's protocol. The viral markers, including hepatitis B surface antigen (HBsAg), anti-hepatitis B s antibody (HBsAb) hepatitis B e antigen (HBeAg), and anti-hepatitis B e antibody (HBeAb), were determined using commercial chemiluminescence immunoassay kits (cat. nos. S10980090, S10980089, S10980088 and S10980087, respectively; Beijing Wantai Biological Pharmacy Enterprise Co., Ltd., Beijing, China) on an ARCHITECT i-20000SR automatic chemiluminescence immunoassay analyzer (Abbott Laboratories, Chicago, IL, USA). Serum HBV DNA levels were determined using a Cobas HBV Amplicor Monitor assay (Roche Diagnostics, Pleasanton, CA, USA), with a lower limit of detection of 2.46 log10 copies/ml (~50 IU/ml or 291 copies/ml), according to the manufacturer's protocol.
3
Quantitative Assessment of Hepatitis B Viral Markers
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Serum HBsAg was quantified by Elecsys HBsAg II Quant reagent kits (Roche Diagnostics) with a lower limit of detection (LLD) of 0.05 IU/mL. Quantitative levels of HBcrAg were determined by chemiluminescent enzyme immunoassay in an automated analyzer system (Fujirebio Inc., Tokyo, Japan) with the LLD of 1,000 IU/mL and a linear range of 3-7 log IU/mL. Serum HBV RNA was isolated with the nucleic acid extraction or purification kit (Sansure Biotech, Changsha, China) and treated with DNase I (Thermo Fisher Scientific, Waltham, MA, USA). The LLD of the assay was 200 copies/mL. Details on HBV RNA assay can be found in Supplementary Materials
4
Routine Serum Biochemical Markers for HBV
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Routine serum biochemical parameters, including ALT and aspartate aminotransferase (AST) levels, were measured using automated techniques (HITACHI 7600, Japan) (upper limits of normal: 50 IU/L and 40 IU/L, resp.). HBV markers (HBsAg, hepatitis B e antigen (HBeAg), anti-HBe, and anti-HBc) were detected using a commercial chemiluminescent microparticle immunoassay (CMIA) kit with the Architect i2000 system (Abbott Laboratories, USA).
Serum HBV DNA levels were quantified at all time points using the Cobas HBV Amplicor Monitor assay (Roche Diagnostics, Branchburg, NJ, USA) according to the manufacturer instructions in the reagent kit. The detection limit of the assay was 300 viral genome copies/mL (2.48 log10 copies/mL).
Serum HBV DNA levels were quantified at all time points using the Cobas HBV Amplicor Monitor assay (Roche Diagnostics, Branchburg, NJ, USA) according to the manufacturer instructions in the reagent kit. The detection limit of the assay was 300 viral genome copies/mL (2.48 log10 copies/mL).
5
Biomarker Assessments in Hepatitis B Patients
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ALT and aspartate aminotransferase (AST) levels were measured using kits purchased from Shanghai Kehua Bio-Engineering Co., Ltd. (Shanghai, China) and an Olympus Automatic Biochemical Analyzer (AU5400; Olympus Corporation, Tokyo, Japan) with a cut-off value of 40 IU/L. The levels of viral markers, including hepatitis B surface antigen (HBsAg), hepatitis B e-antigen (HBeAg) and antibody against HBeAg (anti-HBe) were determined using commercial chemiluminescent immunoassay kits (Beijing Wantai Biological Pharmacy, Beijing, China) on an ARCHITECT i-20000SR automatic chemiluminescence immunoassay analyzer purchased from Abbott Laboratories (Chicago, IL, USA).
The serum HBV DNA level was determined using the Cobas HBV Amplicor Monitor assay (Roche Molecular Diagnostics, Pleasanton, CA, USA) at baseline, then every 6 months during the first year of therapy and annually for the remaining of the treatment. The lower limit of quantification was 50 IU/ml or 291 copies/ml. From the second year of treatment, the HBV DNA levels were assessed using pyrosequencing (PyroMark Q24 Mdx system; Qiagen GmbH, Hilden, Germany) at 18, 30, 42 and 54 months of follow-up.
The serum HBV DNA level was determined using the Cobas HBV Amplicor Monitor assay (Roche Molecular Diagnostics, Pleasanton, CA, USA) at baseline, then every 6 months during the first year of therapy and annually for the remaining of the treatment. The lower limit of quantification was 50 IU/ml or 291 copies/ml. From the second year of treatment, the HBV DNA levels were assessed using pyrosequencing (PyroMark Q24 Mdx system; Qiagen GmbH, Hilden, Germany) at 18, 30, 42 and 54 months of follow-up.
6
Perinatal Hepatitis B Screening
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Maternal HBV DNA was tested before delivery; HBsAg, anti‐HBs, hepatitis B e antigen (HBeAg), anti‐HBe, anti‐HBc and HBV DNA of infants were tested at the age of 7‐12 months. For HBsAg‐positive mothers and their infants, alanine aminotransferase (ALT), aspartate transaminase (AST), total bilirubin (TBIL), blood glucose (GLU), cholinesterase (CHE) and cholesterol (CHOL) were measured using an Olympus Automatic Biochemical Analyzer AU5400 (Olympus). HBV seromarkers were performed by chemiluminescence methods. The level of HBV DNA was examined using the Cobas HBV Amplicor Monitor assay (Roche Diagnostics). Nationality of Han was the major ethnic group in China.
7
Hepatitis B Serological and Viral Markers
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Hepatitis B surface antigen (HBsAg), hepatitis B e antigen (HBeAg), hepatitis B surface antibody (anti-HBs), hepatitis B e antibody (anti-HBe), and hepatitis B core antibody (anti-HBc) were detected using a commercial Chemiluminescent Microparticle Immunoassay (CMIA) kit with the Architect-i2000 system (Abbott Laboratories, Abbott Park, IL). Serum HBV-DNA was quantified using the Cobas HBV Amplicor Monitor assay (Roche Diagnostics, Branchburg, NJ) according to the instructions of the manufacturer. The lower HBV DNA detection limit was 2.48 log10 copies/mL. Serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were detected using a HITACHI 7600–110 auto biochemical analysis system (Hitachi Ltd., Tokyo, Japan), and the normal range of ALT and AST was 5 to 50 IU/L and 8 to 40 IU/L, respectively.
8
Hepatitis B Viral Load Quantification
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Laboratory test results for ALT, HBsAg, HBsAb, HBeAg, HBeAb and HBV DNA were captured from the HIS during the follow-up period. Serum HBV DNA quantification was determined using Applied Biosystems Real-time PCR system (Prism 7500; Applied Biosystems, Inc., USA, with a low limit of 500 IU/ml) or Roche COBAS HBV Amplicor MonitorTM assay (Roche Diagnostics, Basel, Switzerland; with a low limit of 20 IU/mL). Serum HBsAg, HBsAb, HBeAg and HBeAb were determined using commercial enzyme immunoassay kits (AXSYM System; Abbott, Wiesbaden, Germany). HBsAg was quantified by Abbott Architect HBsAg Reagent Kit (Abbott Ireland Diagnostics Division, Sligo, Ireland; dynamic range 0.05–250.0 IU/ml).
9
Transient Transfection of HBV DNA Plasmid
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HBV DNA plasmid (pHBV), which contains the 1.3mer overlength HBV genome, and backbone plasmid pUC19 were a kind gift of professor James Ou from University of Southern California, Los Angeles, CA (14) .
Transient transfection into HepG2 of pHBV (pHepG2) or the backbone plasmid pUC19 (as the mock-transfected HepG2) were performed using Fugene 6 reagent (Roche) according to the manufacturer's instructions, respectively. HepG2 cells were lysed for both protein and RNA analysis after 48 h transfection. Meanwhile, level of HBsAg in the cell supernatant was determined using the ARCHITECT i2000SR HBsAg QT assay (Abbott). The cut-off value for determining if HBsAg is positive is S/N ratio ≥ 0.05, and the reporting units are S/N ratio. Level of HBV DNA in the cell supernatant was detected using the Roche COBAS HBV Amplicor MonitorTM assay. Tandem mRFP-GFP-LC3 expressing plasmid ptfLC3 (Addgene) was obtained from Addgene (15) , and transfection was performed using Lipofectamine 2000 (Invitrogen).
Transient transfection into HepG2 of pHBV (pHepG2) or the backbone plasmid pUC19 (as the mock-transfected HepG2) were performed using Fugene 6 reagent (Roche) according to the manufacturer's instructions, respectively. HepG2 cells were lysed for both protein and RNA analysis after 48 h transfection. Meanwhile, level of HBsAg in the cell supernatant was determined using the ARCHITECT i2000SR HBsAg QT assay (Abbott). The cut-off value for determining if HBsAg is positive is S/N ratio ≥ 0.05, and the reporting units are S/N ratio. Level of HBV DNA in the cell supernatant was detected using the Roche COBAS HBV Amplicor MonitorTM assay. Tandem mRFP-GFP-LC3 expressing plasmid ptfLC3 (Addgene) was obtained from Addgene (15) , and transfection was performed using Lipofectamine 2000 (Invitrogen).
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