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

Joe Grove, Ke Hu, Michelle J. Farquhar, Margaret Goodall, Lucas Walker, Mohammed Jamshad, Heidi E. Drummer, Roslyn M. Bill, Peter Balfe, Jane A. McKeating

Research output: Contribution to journalArticle

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.

Original languageEnglish
Pages (from-to)82
JournalWellcome Open Research
Volume2
Early online date7 Sep 2017
DOIs
Publication statusPublished - 7 Sep 2017

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Viruses
Hepacivirus
Epitopes
Tetraspanins
Monoclonal Antibodies
Antibodies
Electron Scanning Microscopy
Physiological Phenomena
Epitope Mapping
Virus Internalization
Scanning electron microscopy
Virus Diseases
Pathologic Processes
Eukaryota
Cell Movement
Actins
Anti-Idiotypic Antibodies
Proteins
Chemical activation
Association reactions

Bibliographical note

Funding: BBSRC (BB/N007417/1); EC (LSHG-CT-2004-504601 and FP7 F3-2012-305578); MRC (MC_UU_12018/1 and G1100247); Wellcome Trust (200838); and Royal Society (107653).

Cite this

Grove, J., Hu, K., Farquhar, M. J., Goodall, M., Walker, L., Jamshad, M., ... McKeating, J. A. (2017). A new panel of epitope mapped monoclonal antibodies recognising the prototypical tetraspanin CD81. Wellcome Open Research, 2, 82. https://doi.org/10.12688/wellcomeopenres.12058.1
Grove, Joe ; Hu, Ke ; Farquhar, Michelle J. ; Goodall, Margaret ; Walker, Lucas ; Jamshad, Mohammed ; Drummer, Heidi E. ; Bill, Roslyn M. ; Balfe, Peter ; McKeating, Jane A. / A new panel of epitope mapped monoclonal antibodies recognising the prototypical tetraspanin CD81. In: Wellcome Open Research. 2017 ; Vol. 2. pp. 82.
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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.",
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Grove, J, Hu, K, Farquhar, MJ, Goodall, M, Walker, L, Jamshad, M, Drummer, HE, Bill, RM, Balfe, P & McKeating, JA 2017, 'A new panel of epitope mapped monoclonal antibodies recognising the prototypical tetraspanin CD81', Wellcome Open Research, vol. 2, pp. 82. https://doi.org/10.12688/wellcomeopenres.12058.1

A new panel of epitope mapped monoclonal antibodies recognising the prototypical tetraspanin CD81. / Grove, Joe; Hu, Ke; Farquhar, Michelle J.; Goodall, Margaret; Walker, Lucas; Jamshad, Mohammed; Drummer, Heidi E.; Bill, Roslyn M.; Balfe, Peter; McKeating, Jane A.

In: Wellcome Open Research, Vol. 2, 07.09.2017, p. 82.

Research output: Contribution to journalArticle

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T1 - A new panel of epitope mapped monoclonal antibodies recognising the prototypical tetraspanin CD81

AU - Grove, Joe

AU - Hu, Ke

AU - Farquhar, Michelle J.

AU - Goodall, Margaret

AU - Walker, Lucas

AU - Jamshad, Mohammed

AU - Drummer, Heidi E.

AU - Bill, Roslyn M.

AU - Balfe, Peter

AU - McKeating, Jane A.

N1 - Funding: BBSRC (BB/N007417/1); EC (LSHG-CT-2004-504601 and FP7 F3-2012-305578); MRC (MC_UU_12018/1 and G1100247); Wellcome Trust (200838); and Royal Society (107653).

PY - 2017/9/7

Y1 - 2017/9/7

N2 - 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.

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Grove J, Hu K, Farquhar MJ, Goodall M, Walker L, Jamshad M et al. A new panel of epitope mapped monoclonal antibodies recognising the prototypical tetraspanin CD81. Wellcome Open Research. 2017 Sep 7;2:82. https://doi.org/10.12688/wellcomeopenres.12058.1