Pelletier Julie, Salem Mabrouka, Lecka Joanna, Fausther Michel, Bigonnesse François, Sévigny Jean
Centre de recherche du CHU de Québec - Université Laval, Québec City QC, Canada.
Centre de recherche du CHU de Québec - Université Laval, Québec CityQC, Canada; Département de Microbiologie-Infectiologie et d'Immunologie, Faculté de Médecine, Université Laval, Québec CityQC, Canada.
Front Pharmacol. 2017 Mar 8;8:115. doi: 10.3389/fphar.2017.00115. eCollection 2017.
The ectonucleotidase nucleoside triphosphate diphosphohydrolase-8 (NTPDase8) is the last member of the Ecto-NTPDase family to be discovered and characterized. It is a transmembrane protein which regulates the concentration of the agonists of P1 and P2 receptors at the cell surface. The functions of the enzyme are still not known partly due to the lack of specific tools such as antibodies. In this work, guinea pig polyclonal antibodies against mouse NTPDase8 and mouse monoclonal antibodies against human NTPDase8 have been generated and characterized. For the production of antibodies against mouse NTPDase8 several techniques have been tried. Several peptide antigens in several hosts (rabbit, rat, hamster, and guinea pig) failed to give a positive reaction suggesting that NTPDase8 is poorly immunogenic. In this study, we describe the successful process that led to anti-mouse NTPDase8, namely the cDNA immunization technique. Monoclonal antibodies to human NTPDase8 were also obtained by cDNA immunization followed by a final injection with transfected human embryonic kidney (HEK 293T) cells expressing human NTPDase8. The specificity of these antibodies was evaluated by Western blot, immunocytochemistry, immunohistochemistry and flow cytometry. In contrast, all commercial antibodies to NTPDase8 peptides that we have tested failed to give a specific positive signal against the expressed NTPDase8 protein when used to probe Western blots. In addition, immunohistochemistry experiments confirmed the presence of NTPDase8 in mouse liver canaliculi. The tools generated in this work will help characterize NTPDase8 localization and function in future studies and its contribution to the modulation of P1 and P2 receptor activation.
胞外核苷酸酶核苷三磷酸二磷酸水解酶8（NTPDase8）是胞外NTPDase家族中最后一个被发现和鉴定的成员。它是一种跨膜蛋白，可调节细胞表面P1和P2受体激动剂的浓度。由于缺乏抗体等特异性工具，该酶的功能仍不清楚。在这项工作中，已经制备并鉴定了针对小鼠NTPDase8的豚鼠多克隆抗体和针对人NTPDase8的小鼠单克隆抗体。为了制备针对小鼠NTPDase8的抗体，尝试了几种技术。在几种宿主（兔、大鼠、仓鼠和豚鼠）中使用几种肽抗原均未产生阳性反应，这表明NTPDase8的免疫原性较差。在本研究中，我们描述了成功制备抗小鼠NTPDase8抗体的过程，即cDNA免疫技术。通过cDNA免疫，然后用表达人NTPDase8的转染人胚肾（HEK 293T）细胞进行最后一次注射，也获得了针对人NTPDase8的单克隆抗体。通过蛋白质印迹、免疫细胞化学、免疫组织化学和流式细胞术评估了这些抗体的特异性。相比之下，我们测试的所有针对NTPDase8肽的商业抗体在用于探测蛋白质印迹时，均未针对表达的NTPDase8蛋白产生特异性阳性信号。此外，免疫组织化学实验证实了NTPDase8在小鼠肝小管中的存在。这项工作中产生的工具将有助于在未来的研究中鉴定NTPDase8的定位和功能，以及其对P1和P2受体激活调节的贡献。
