Epitope Mapping of an Anti-elephant Podoplanin Monoclonal Antibody (PMab-295) Using Enzyme-Linked Immunosorbent Assay
Epitope Mapping of an Anti-elephant Podoplanin Monoclonal Antibody (PMab-295) Using Enzyme-Linked Immunosorbent Assay
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使用酶联免疫吸附测定法对抗大象 Podoplanin 单克隆抗体 (PMab-295) 进行表位作图
DOI：
10.1089/mab.2022.0017
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发表时间：
2022
影响因子：
--
通讯作者：
Kato Yukinari
中科院分区：
文献类型：
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作者：
Okada Yuki;Suzuki Hiroyuki;Kaneko Mika K.;Kato Yukinari
Podoplanin (PDPN) 是肺 I 型肺泡细胞、肾足细胞和淋巴管内皮细胞的标记物。 PDPN的过度表达有助于肿瘤的恶性进展。因此，开发动物抗 PDPN 单克隆抗体 (mAb) 对于评估发病机制和细胞功能至关重要。我们之前利用肽免疫开发了一种抗大象 PDPN (elePDPN) mAb PMab-295，可用于流式细胞术、蛋白质印迹和免疫组织化学。在本研究中，我们通过酶联免疫吸附测定 (ELISA) 确定了 PMab-295 的关键表位。我们对 elePDPN 胞外域（氨基酸 38-51）的丙氨酸取代肽进行 ELISA，发现 PMab-295 不识别 M41A、P44A 和 E47A 的丙氨酸取代肽。此外，通过流式细胞术和免疫组织化学，这些肽不能抑制PMab-295对表达elePDPN的细胞的识别。结果表明PMab-295的结合表位包括elePDPN的Met41、Pro44和Glu47。
Podoplanin (PDPN) is a marker of lung type I alveolar cells, kidney podocytes, and lymphatic endothelial cells. The overexpression of PDPN contributes to the malignant progression of tumors. Therefore, the development of anti-PDPN monoclonal antibodies (mAbs) to animals is essential to evaluate the pathogenesis and cellular functions. Using peptide immunization, we previously developed an anti-elephant PDPN (elePDPN) mAb, PMab-295, which is useful for flow cytometry, Western blotting, and immunohistochemistry. In this study, we determined the critical epitope of PMab-295 by enzyme-linked immunosorbent assay (ELISA). We performed ELISA with the alanine-substituted peptides of elePDPN extracellular domain (amino acids 38–51), and found that PMab-295 did not recognize the alanine-substituted peptides of M41A, P44A, and E47A. Furthermore, these peptides could not inhibit the recognition of PMab-295 to elePDPN-expressing cells by flow cytometry and immunohistochemistry. The results indicate that the binding epitope of PMab-295 includes Met41, Pro44, and Glu47 of elePDPN.