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Wellcome Open Res. 2017 Sep 7;2:82. doi: 10.12688/wellcomeopenres.12058.1. eCollection 2017.

A new panel of epitope mapped monoclonal antibodies recognising the prototypical tetraspanin CD81.

Author information

1
Institute of Immunity and Transplantation, Division of Infection and Immunity, , University College London, London, NW3 2PF, UK.
2
Institute of Immunology and Immunotherapy, University of Birmingham, Birmingham, B15 2TT, UK.
3
Institute for Microbiology and Infection, School of Biosciences, University of Birmingham, Birmingham, B15 2TT, UK.
4
Centre for Biomedical Resear, Burnet Institute, Melbourne, VIC, 3004, Australia.
5
School of Life and Health Sciences, Aston University, Birmingham, B4 7ET, UK.
6
Nuffield Department of Medicine, University of Oxford, Oxford, OX3 7BN, UK.
#
Contributed equally

Abstract

Background: Tetraspanins are small transmembrane proteins, found in all higher eukaryotes, that compartmentalize cellular membranes through interactions with partner proteins. CD81 is a prototypical tetraspanin and contributes to numerous physiological and pathological processes, including acting as a critical entry receptor for hepatitis C virus (HCV). Antibody engagement of tetraspanins can induce a variety of effects, including actin cytoskeletal rearrangements, activation of MAPK-ERK signaling and cell migration. However, the epitope specificity of most anti-tetraspanin antibodies is not known, limiting mechanistic interpretation of these studies. Methods: We generated a panel of monoclonal antibodies (mAbs) specific for CD81 second extracellular domain (EC2) and performed detailed epitope mapping with a panel of CD81 mutants. All mAbs were screened for their ability to inhibit HCV infection and E2-CD81 association. Nanoscale distribution of cell surface CD81 was investigated by scanning electron microscopy. Results: The antibodies were classified in two epitope groups targeting opposing sides of EC2. We observed a wide range of anti-HCV potencies that were independent of their epitope grouping, but associated with their relative affinity for cell-surface expressed CD81. Scanning electron microscopy identified at least two populations of CD81; monodisperse and higher-order assemblies, consistent with tetraspanin-enriched microdomains. Conclusions: These novel antibodies provide well-characterised tools to investigate CD81 function, including HCV entry, and have the potential to provide insights into tetraspanin biology in general.

KEYWORDS:

CD81; hepatitis C virus; tetraspanin

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