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Development of Antibody and Nanobody Tools for P2X7

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The P2X7 Receptor

Part of the book series: Methods in Molecular Biology ((MIMB,volume 2510))

Abstract

Antibodies that recognize the ATP-gated P2X7 ion channel are etablished research tools. Nanobodies correspond to the antigen-binding variable immunoglobulin domain (VHH) of heavy chain antibodies that naturally occur in camelids. Nanobodies display better solubility than the variable domains (VH) of conventional antibodies. Therefore, it is much easier to construct bivalent and multivalent fusion proteins with nanobodies than with VH domains or with paired VH-VL domains. Moreover, nanobodies can bind functional crevices that are poorly accessbile to conventional VH-VL domains. This makes nanobodies particulary well suited as functional modulators. Here we provide protocols to raise antibodies and nanobodies against mouse and human P2X7 using cDNA-immunization. This approach evokes antibodies and nanobodies that recognize the P2X7 ion channel in native confirmation, some of which inhibit or potentiate gating of P2X7 by extracellular ATP. Furthermore, we developed protocols for producing P2X7-specific nanobodies and antibodies in vivo using rAAV vectors (AAVnano). This approach can be used either to durably inhibit or potentiate P2X7 function in vivo, or to deplete P2X7-expressing cells.

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Acknowledgments

Supported by grants from the Deutsche Forschungsgemeinschaft to F.H. and F.K.-N (No310/13-1, SFB1328A10 and SFB1328Z02), and from the Agence Nationale de la Recherche to S.A. (ANR-18-CE92-0046). We thank Fabienne Seyfried, Gudrun Dubberke, Anna Josephine Gebhardt, and Dorte Wendt (Institute of Immunology, UKE, Hamburg) and Sarah Hewald, Nicole Lüder and Andrea Nitsche, (UKE central animal facility), for excellent technical help with antibody cloning and immunizations.

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Author notes

  1. Sahil Adriouch, Friedrich Koch-Nolte, Stephan Menzel share senior authorship.

Authors and Affiliations

  1. Institute of Immunology, University Medical Center Hamburg-Eppendorf, Hamburg, Germany

    Tobias Stähler, Welbeck Danquah, Friedrich Haag, Friedrich Koch-Nolte & Stephan Menzel

  2. UNIROUEN, INSERM, U1234, Pathophysiology, Autoimmunity, aNd immunoTHERapies (PANTHER), Normandie University, 76000 Rouen, France

    Melanie Demeules, Henri Gondé, Romain Hardet & Sahil Adriouch

Authors
  1. Tobias Stähler
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  2. Welbeck Danquah
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  3. Melanie Demeules
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  4. Henri Gondé
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  5. Romain Hardet
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  6. Friedrich Haag
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  7. Sahil Adriouch
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  8. Friedrich Koch-Nolte
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  9. Stephan Menzel
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Corresponding author

Correspondence to Friedrich Koch-Nolte .

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Editors and Affiliations

  1. Walther Straub Institute of Pharmacology and Toxicology, Ludwig-Maximilians-Universität München, Munich, Germany

    Annette Nicke

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Stähler, T. et al. (2022). Development of Antibody and Nanobody Tools for P2X7. In: Nicke, A. (eds) The P2X7 Receptor. Methods in Molecular Biology, vol 2510. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-2384-8_6

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  • DOI: https://doi.org/10.1007/978-1-0716-2384-8_6

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  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-0716-2383-1

  • Online ISBN: 978-1-0716-2384-8

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