Honma Ryusuke, Fujii Yuki, Ogasawara Satoshi, Oki Hiroharu, Konnai Satoru, Kagawa Yumiko, Takagi Michiaki, Kaneko Mika K, Kato Yukinari
1 Department of Regional Innovation, Tohoku University Graduate School of Medicine , Sendai, Japan .
2 Department of Orthopaedic Surgery, Yamagata University Faculty of Medicine , Yamagata, Japan .
Monoclon Antib Immunodiagn Immunother. 2016 Apr;35(2):65-72. doi: 10.1089/mab.2015.0078. Epub 2016 Mar 15.
Podoplanin (PDPN) is a type I transmembrane sialoglycoprotein, which is expressed in several normal cells, including lymphatic endothelial cells throughout the body, podocytes of the kidney, and lung type I alveolar cells of the lung. We have established many monoclonal antibodies (mAbs) against human PDPN, mouse PDPN, and rat PDPN. In addition, we recently produced an anti-rabbit PDPN mAb, PMab-32, which was established by immunizing mice with recombinant proteins of rabbit PDPN. Herein, we compared the reactivity of PMab-32 with that of newly established anti-rabbit PDPN mAbs, PMab-33 and PMab-21, against normal tissues in immunohistochemistry. PMab-32 reacted with podocytes, type I alveolar cells, and lymphatic endothelial cells, whereas PMab-33 detected only podocytes and type I alveolar cells but not lymphatic endothelial cells. PMab-21 was not useful in immunohistochemistry. We identified the epitope of PMab-32 and PMab-33 as Ser61-Ala68 of rabbit PDPN using western blot and flow cytometric analyses. In contrast, the epitope of PMab-21 was identified as Leu44-Glu48, which is corresponding to platelet aggregation-stimulating (PLAG) domain, indicating that Ser61-Ala68 of rabbit PDPN is a more appropriate epitope for immunohistochemistry compared with PLAG domain. PMab-32 could be useful for uncovering the function of rabbit PDPN.
血小板内皮细胞黏附分子（PDPN）是一种I型跨膜唾液酸糖蛋白，在多种正常细胞中表达，包括全身的淋巴管内皮细胞、肾足细胞和肺I型肺泡细胞。我们已经制备了许多针对人PDPN、小鼠PDPN和大鼠PDPN的单克隆抗体（mAb）。此外，我们最近制备了一种抗兔PDPN单克隆抗体PMab - 32，它是通过用兔PDPN重组蛋白免疫小鼠而制备的。在此，我们在免疫组织化学中比较了PMab - 32与新制备的抗兔PDPN单克隆抗体PMab - 33和PMab - 21对正常组织的反应性。PMab - 32与足细胞、I型肺泡细胞和淋巴管内皮细胞发生反应，而PMab - 33仅检测到足细胞和I型肺泡细胞，未检测到淋巴管内皮细胞。PMab - 21在免疫组织化学中没有用处。我们使用蛋白质印迹法和流式细胞术分析确定了PMab - 32和PMab - 33的表位为兔PDPN的Ser61 - Ala68。相比之下，PMab - 21的表位被确定为Leu44 - Glu48，其对应于血小板聚集刺激（PLAG）结构域，这表明与PLAG结构域相比，兔PDPN的Ser61 - Ala68是免疫组织化学中更合适的表位。PMab - 32可能有助于揭示兔PDPN的功能。
