Development and Characterization of Anti-Sheep Podoplanin Monoclonal Antibodies PMab-253 and PMab-260
Development and Characterization of Anti-Sheep Podoplanin Monoclonal Antibodies PMab-253 and PMab-260
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抗绵羊足足蛋白单克隆抗体 PMab-253 和 PMab-260 的开发和表征
DOI：
10.1089/mab.2020.0018
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发表时间：
2020
期刊：
影响因子：
--
通讯作者：
Kato Y.
中科院分区：
文献类型：
--
作者：
Kaneko MK;Sano M;Takei J;Asano T;Sayama Y;Hosono H;Kobayashi A;Konnai S;Kato Y.
在免疫组织化学分析中，需要抗podoplanin（PDPN）单克隆抗体（mAb）作为淋巴管内皮细胞或I型肺泡细胞的标记物。我们已经开发了用于许多物种的抗PDPN mAb，包括人、小鼠、大鼠、兔、狗、猫、牛、猪、塔斯马尼亚魔鬼、羊驼、老虎、鲸鱼、山羊、马和熊。本研究采用基于细胞的免疫和筛选（CBIS）方法开发和鉴定抗绵羊PDPN（sPDPN）的mAb。将RAP 14标签添加到sPDPN的N-末端，并使用抗RAP 14标签mAb（PMab-2）在流式细胞术和Western印迹中测量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抗sPDPN的mAb。其中两种mAb PMab-253（免疫球蛋白M [IgM]，κ）和PMab-260（IgM，κ）使用流式细胞术和蛋白质印迹法特异性检测CHO/sPDPN细胞。PMab-253和PMab-260均对肾小球和Bowman囊、肺和结肠的淋巴管内皮细胞以及肺的I型肺泡细胞进行染色，表明通过CBIS方法开发的PMab-253和PMab-260可用于sPDPN的功能分析。我们还使用流式细胞术确定PMab-253和PMab-260的结合表位。对sPDPN缺失突变体的分析显示，PMab-253和PMab-260表位的N-末端存在于sPDPN的氨基酸110和115之间。对sPDPN点突变的分析显示，PMab-253和PMab-260的关键表位包括sPDPN的Thr 112和Ser 113，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。
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.