Institute of Immunology, University Medical Center Hamburg-Eppendorf, Hamburg, Germany.
UNIROUEN, INSERM, U1234, Pathophysiology, Autoimmunity, aNd immunoTHERapies (PANTHER), Normandie University, 76000 Rouen, France.
Methods Mol Biol. 2022;2510:99-127. doi: 10.1007/978-1-0716-2384-8_6.
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.
能够识别三磷酸腺苷门控 P2X7 离子通道的抗体是已建立的研究工具。纳米抗体对应于重链抗体的抗原结合可变免疫球蛋白结构域（VHH），它在骆驼科动物中自然存在。纳米抗体比传统抗体的可变结构域（VH）具有更好的溶解性。因此，与 VH 结构域或配对的 VH-VL 结构域相比，用纳米抗体构建二价和多价融合蛋白要容易得多。此外，纳米抗体可以结合传统 VH-VL 结构域难以进入的功能裂隙。这使得纳米抗体特别适合作为功能性调节剂。在这里，我们提供了使用 cDNA 免疫来针对小鼠和人 P2X7 产生抗体和纳米抗体的方案。这种方法可以产生识别天然构象中 P2X7 离子通道的抗体和纳米抗体，其中一些可以抑制或增强细胞外 ATP 对 P2X7 的门控作用。此外，我们还开发了使用 rAAV 载体（AAVnano）在体内产生 P2X7 特异性纳米抗体和抗体的方案。该方法可用于在体内持久抑制或增强 P2X7 功能，或耗尽表达 P2X7 的细胞。
