Department of Antibody Drug Development, Tohoku University Graduate School of Medicine, Sendai, Japan.
Laboratory of Comparative Pathology, Faculty of Veterinary Medicine, Hokkaido University, Sapporo, Japan.
Monoclon Antib Immunodiagn Immunother. 2020 Aug;39(4):144-155. doi: 10.1089/mab.2020.0018. Epub 2020 Jul 16.
Anti-podoplanin (PDPN) monoclonal antibodies (mAbs) are needed as markers for lymphatic endothelial cells or type I alveolar cells in immunohistochemical analyses. We have developed anti-PDPN mAbs for many species, including humans, mice, rats, rabbits, dogs, cats, bovines, pigs, Tasmanian devils, alpacas, tigers, whales, goats, horses, and bears. This study develops and characterizes anti-sheep PDPN (sPDPN) mAbs using Cell-Based Immunization and Screening (CBIS) method. A RAP14 tag was added to the N-terminus of sPDPN, and an anti-RAP14 tag mAb (PMab-2) was used to measure the expression level of sPDPN in flow cytometry and Western blots. We immunized mice with sPDPN-overexpressing Chinese hamster ovary (CHO)-K1 (CHO/sPDPN) cells and screened mAbs against sPDPN using flow cytometry. Two of the mAbs, PMab-253 (immunoglobulin M [IgM], kappa) and PMab-260 (IgM, kappa), detected CHO/sPDPN cells specifically using flow cytometry and Western blots. Both PMab-253 and PMab-260 stained the renal glomerulus and Bowman's capsule, lymphatic endothelial cells of the lung and colon, and type I alveolar cells of the lung, suggesting PMab-253 and PMab-260, which were developed by CBIS method, can be applied to functional analyses of sPDPN. We also determined the binding epitope of PMab-253 and PMab-260 using flow cytometry. Analysis of sPDPN deletion mutants revealed that the N-terminus of the PMab-253 and PMab-260 epitope exists between amino acids 110 and 115 of sPDPN. Analysis of sPDPN point mutations revealed that the critical epitope of PMab-253 and PMab-260 includes Thr112 and Ser113 of sPDPN, indicating that the PMab-253 and PMab-260 epitope are independent of the platelet aggregation-stimulating (PLAG) domain or the PLAG-like domain of sPDPN.
抗 podoplanin (PDPN) 单克隆抗体 (mAbs) 是免疫组织化学分析中用于标记淋巴管内皮细胞或 I 型肺泡细胞的必要标志物。我们已经为许多物种开发了抗 PDPN mAbs，包括人类、小鼠、大鼠、兔子、狗、猫、牛、猪、塔斯马尼亚恶魔、羊驼、老虎、鲸鱼、山羊、马和熊。本研究使用基于细胞的免疫接种和筛选 (CBIS) 方法开发和表征抗绵羊 PDPN (sPDPN) mAbs。在 sPDPN 的 N 端添加了一个 RAP14 标签，并用抗 RAP14 标签 mAb (PMab-2) 测量流式细胞术和 Western blot 中 sPDPN 的表达水平。我们用过表达 sPDPN 的中国仓鼠卵巢 (CHO)-K1 (CHO/sPDPN) 细胞免疫小鼠，并使用流式细胞术筛选针对 sPDPN 的 mAbs。两种 mAbs，PMab-253(免疫球蛋白 M[IgM]，kappa)和 PMab-260(IgM，kappa)，通过流式细胞术和 Western blot 特异性检测 CHO/sPDPN 细胞。PMab-253 和 PMab-260 均染色肾脏肾小球和鲍曼氏囊、肺和结肠的淋巴管内皮细胞以及肺的 I 型肺泡细胞，表明通过 CBIS 方法开发的 PMab-253 和 PMab-260 可应用于 sPDPN 的功能分析。我们还使用流式细胞术测定了 PMab-253 和 PMab-260 的结合表位。通过 sPDPN 缺失突变体分析，PMab-253 和 PMab-260 的表位存在于 sPDPN 的第 110 至 115 位氨基酸之间。sPDPN 点突变分析表明，PMab-253 和 PMab-260 的关键表位包括 sPDPN 的 Thr112 和 Ser113，表明 PMab-253 和 PMab-260 表位不依赖于 sPDPN 的血小板聚集刺激 (PLAG) 结构域或 PLAG 样结构域。
