Kato Yukinari, Furusawa Yoshikazu, Yamada Shinji, Itai Shunsuke, Takei Junko, Sano Masato, Kaneko Mika K
New Industry Creation Hatchery Center, Tohoku University, 2-1 Seiryo-machi, Aoba-ku, Sendai, Miyagi, 980-8575, Japan.
Department of Antibody Drug Development, Tohoku University Graduate School of Medicine, 2-1 Seiryo-machi, Aoba-ku, Sendai, Miyagi, 980-8575, Japan.
Biochem Biophys Rep. 2019 Apr 5;18:100633. doi: 10.1016/j.bbrep.2019.100633. eCollection 2019 Jul.
Podoplanin (PDPN) is known as a lymphatic endothelial cell marker. Monoclonal antibodies (mAbs) against human, mouse, rat, rabbit, dog, cat, bovine, pig, and horse PDPN have been established in our previous studies. However, mAbs against alpaca PDPN (aPDPN), required for immunohistochemical analysis, remain to be developed. In the present study, we employed the Cell-Based Immunization and Screening (CBIS) method for producing -aPDPN mAbs. We immunized mice with aPDPN-overexpressing Chinese hamster ovary (CHO)-K1 cells (CHO/aPDPN), and hybridomas producing mAbs against aPDPN were screened using flow cytometry. One of the mAbs, PMab-225 (IgG, kappa), specifically detected CHO/aPDPN cells via flow cytometry and recognized the aPDPN protein on Western blotting. Further, PMab-225 strongly stained lung type I alveolar cells, colon lymphatic endothelial cells, and kidney podocytes via immunohistochemistry. These findings demonstrate that PMab-225 antibody is useful to investigate the function of aPDPN via different techniques.
血小板反应蛋白-1结构域蛋白（PDPN）是一种淋巴管内皮细胞标志物。在我们之前的研究中，已经制备了针对人、小鼠、大鼠、兔、狗、猫、牛、猪和马PDPN的单克隆抗体（mAb）。然而，用于免疫组织化学分析所需的抗羊驼PDPN（aPDPN）单克隆抗体仍有待开发。在本研究中，我们采用基于细胞的免疫和筛选（CBIS）方法来制备抗aPDPN单克隆抗体。我们用过量表达aPDPN的中国仓鼠卵巢（CHO）-K1细胞（CHO/aPDPN）免疫小鼠，并使用流式细胞术筛选产生抗aPDPN单克隆抗体的杂交瘤。其中一种单克隆抗体PMab-225（IgG，κ），通过流式细胞术特异性检测CHO/aPDPN细胞，并在蛋白质印迹法中识别aPDPN蛋白。此外，PMab-225通过免疫组织化学强烈染色肺I型肺泡细胞、结肠淋巴管内皮细胞和肾足细胞。这些发现表明，PMab-225抗体可通过不同技术用于研究aPDPN的功能。
