HBV Research Protocols

There is an urgent need for centralized repositories of HBV-related materials that are readily accessible to HBV researchers globally. Critical to this will be quality assurance of the samples, and the availability of matching clinical data. This repository of HBV-related research protocols is designed to facilitate studies and the development of new drugs.

This project is designed to complement the upcoming NIAID reagents repository by making corresponding quality-controlled research protocols available freely for all researchers around the world.

When citing protocols from this database, please cite the original publications from which these protocols have been adapted. The origin of these original publications can be found within the protocols. Please also acknowledge this ICE-HBV Protocols Database. Any questions can be directed to info@ice-hbv.org

The review of these protocols has been led by Haitao Guo and the ICE-HBV working group members including Lena Allweiss, Maura Dandri, Jianming Hu, Jake Liang, Margaret Littlejohn, Peter Revill, and Barbara Testoni.

The development of the database is coordinated by Marley Easterbrook.

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An Optimized Ex Vivo ELISpot assay to identify IFN gamma positive, HBV-specific T cells in Chronic Hepatitis B patients

Conan Chua, Aman Mehrotra and Dr. Adam Gehring

Toronto Center for Liver Disease
Cell Cultures Immunology Assays

During a traditional ELISpot assay, low HBV Specific T cell frequencies have hindered effective ex vivo analysis. We overcame this obstacle to measure ex vivo T cell responses in CHB patients, by modifying the key variables of cell number and the peptide pulsing method to improve ex vivo detection of HBV-specific T cells.

A modified kit-based HBV protein-free DNA extraction from liver tissues and cell cultures for HBV cccDNA Southern blot and qPCR

Mei Yu (1), Maura Dandri (2, 3), Guofeng Cheng* (1), William Delaney* (1), Simon P. Fletcher (1), Lena Allweiss (2, 3)

(1) Gilead Sciences, Foster City, California, United States of America (2) I. Medical Clinic and Polyclinic, University Medical Center Hamburg-Eppendorf, Hamburg, Germany (3) German Center for Infection Research (DZIF), Hamburg-Lübeck-Borstel-Riems Site, Hamburg, Germany
  • cccDNA quantification from total DNA preparation by qPCR with cccDNA-selective primers is challenging because of the coexisting HBV DNA forms (replicative intermediates, protein-free rcDNA (aka deprote... [Read more]

A sensitive and rapid Southern blot assay based on branched DNA technology for the detection of HBV DNA in cell culture and liver tissue samples

Mei Yu (1), Maura Dandri (2, 3), Guofeng Cheng* (1), William Delaney* (1), Simon P. Fletcher (1), Lena Allweiss (2, 3)

(1) Gilead Sciences, Foster City, California, United States of America (2) I. Medical Clinic and Polyclinic, University Medical Center Hamburg-Eppendorf, Hamburg, Germany (3) German Center for Infection Research (DZIF), Hamburg-Lübeck-Borstel-Riems Site, Hamburg, Germany *affiliations at the time of protocol establishment
  • Sothern blot is considered the “gold standard” for cccDNA detection because it can separate the cccDNA from protein-free relaxed circular DNA (pf-rcDNA, aka deproteinated rcDNA (DP-rcD... [Read more]

An Optimized Ex Vivo Flurospot assay to identify multi-functional HBV-specific T cells in Chronic Hepatitis B patients

Conan Chua, Aman Mehrotra, Dr. Adam Gehring

Toronto Centre for Liver Disease, Toronto, Ontario, Canada
Cell Cultures Immunology Assays
During a traditional FluroSpot assay, low HBV Specific T cell frequencies have hindered effective ex vivo analysis. We overcame this obstacle to measure ex vivo T cell responses in CHB patients, by modifying the key variables of cell number and the peptide pulsing method to improve ex vivo detection of HBV-specific T cells.

Fluorescent-bait labelling for the ex vivo detection of HBV antigen-specific B cells

Nina Le Bert (1), Loghman Salimzadeh (1), Alice R. Burton (2), Mala K. Maini (2) Antonio Bertoletti (1)

(1) Emerging Infectious Diseases Program, Duke-NUS Medical School, Singapore (2) Division of Infection and Immunity, UCL, London, UK
Immunology Assays
Fluorescently conjugated antigen-bait systems have been extensively used to identify antigen-specific B cells and probe humoral immunity across different settings. Using this principle, HBV antigens are used to bind the B cell receptor (BCR), permitting an... [Read more]

Tetramer enrichment for HBV-specific CD8+ T cells

Kathrin Heim, Nina Hensel, Maike Hofmann, Rober Thimme

Department of Medicine II, University Hospital Freiburg, Freiburg, Germany
Immunology Assays
The frequency of HBV-specific CD8+ T cells can be very low, depending on the specificity. Therefore, they cannot always be detected by conventional ex vivo tetramer staining using 106 PBMCs. Tetramer-based magnetic enrichment enables the detection of these rare virus-spec... [Read more]

A fluorescent in situ hybridization (FISH) assay for detection of HBV DNA in cell culture models

Xiaonan Zhang, Lei Yue, Chang Li, Zhenghong Yuan

Shanghai Public Health Clinical Center, Key Lab of Medical Molecular Virology, Shanghai Medical College, Fudan University.
Cell Cultures HBV Nucleic Acid Analyses
  • Although a plethora of knowledge on the molecular life cycle of Hepatitis B Virus has been gained by utilizing classical methods, they can only inform on the average level of the tested molecule without subcellular or histological con... [Read more]

HBV Ribonuclease H FRET Assay

Cassandra Kukla, Nathan Ponzar, and John Tavis. Saint Louis University, St. Louis, USA

Saint Louis University
HBV Biochemical Assays
  • This assay detects the activity of HBV RNaseH using fluorescence resonance energy transfer
  • The ribonuclease H (RNaseH) substrate is an RNA DNA heteroduplex in which an RNA oligonucleotide contains a fluorophore and the complementary DNA oligonucleotide contains a fluorescence quencher. ... [Read more]