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 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]

Modeling HBV infection and therapy in immunodeficient NOD-Rag1-/-IL2RgammaC-null (NRG) fumarylacetoacetate hydrolase (FAH) knockout mice with human chimeric liver

James Ahodantin (1), Feng Li (2), and Lishan Su (1)

(1) Lineberger Comprehensive Cancer Center, Department of microbiology and immunology, school of medicine, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA. (2) Guangzhou Eighth People’s Hospital, Guangzhou Medical University, Guangzhou 510060, Guangdong, China. *Correspondence: Correspondence should be addressed to L.S. (lsu@med.unc.edu), telephone: 919-966-6654; fax: 919-966-8212
Animal Models
  • Besides human, only a few species have been reported to be permissive for Hepatitis B virus (HBV) infection(1-3). Human liver chimeric mouse models have been developed to o... [Read more]

Hepatitis B Virus Polymerase Epsilon RNA Binding Assays

Daniel N. Clark, Scott A. Jones, Jianming Hu

Penn State University College of Medicine
HBV Biochemical Assays
  • HBV polymerase activity is assayed by measuring its ability to bind viral RNA at a secondary-structure motif, the epsilon (e) stem-loop structure.
  • HBV polymerase is expressed in cell culture to yield its binding-active form and RNA may be supplied in culture or... [Read more]

In Vitro Hepatitis B Virus Polymerase Priming Assay

Daniel N. Clark, Scott A. Jones, Jianming Hu

Penn State University College of Medicine
HBV Biochemical Assays
  • HBV polymerase activity is assayed by measuring the first step in DNA synthesis, protein priming, wherein the polymerase becomes nucleotide-labelled.
  • This in vitroassay uses a plasmid-based expression system performed in cell culture, producing a chaperone... [Read more]