Development of Antibody and Nanobody Tools for P2X7

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.

Keywords: Heavy chain antibodies; Immunization; Monoclonal antibodies; Nanobodies; P2X7; Phage display; Purinergic signaling; VHH.