Development of Sensitive Monoclonal Antibody PMab-2 Against Rat Podoplanin
Development of Sensitive Monoclonal Antibody PMab-2 Against Rat Podoplanin
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DOI：
10.1089/mab.2015.0041
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发表时间：
2015-12-01
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：
Oki, Hiroharu;Honma, Ryusuke;Kato, Yukinari
Podoplanin（PDPN）是一种血小板聚集诱导因子，是C型凝集素样受体2（CLEC-2）的内源性配体。PDPN也在几种正常组织中表达，如肺I型肺泡细胞和肾足细胞。尽管已经建立了许多抗人PDPN（hPDPN）或小鼠PDPN（mPDPN）的单克隆抗体（MAbs），但尚未开发抗大鼠PDPN（rPDPN）的MAbs，特别是抗rPDPN的血小板聚集刺激（PLAG）结构域（29-54个氨基酸）的MAbs。因此，rPDPN在正常组织中的功能分析受到限制。本研究以rPDPN多肽（38-51个氨基酸）免疫小鼠，制备了一种新的小鼠抗rPDPN单克隆抗体PMab-2（IgG（1），kappa），与抗hPDPN或mPDPN单克隆抗体相比，具有较高的亲和力。测定PMab-2的K-D为5.9 × 10（-10）M。PMab-2不仅可用于针对大鼠真皮成纤维细胞中表达的内源性rPDPN的流式细胞术和Western印迹分析，而且可用于针对正常组织的免疫组织化学。PMab-2在免疫组化中显示出非常高的灵敏度，表明PMab-2对于rPDPN的功能分析非常有利。
Podoplanin (PDPN) is a platelet aggregation-inducing factor, which is known as an endogenous ligand of C-type lectin-like receptor-2 (CLEC-2). PDPN is also expressed in several normal tissues, such as lung type I alveolar cells and kidney podocytes. Although many monoclonal antibodies (MAbs) against human PDPN (hPDPN) or mouse PDPN (mPDPN) have been established, anti-rat PDPN (rPDPN) MAbs, especially against platelet aggregation-stimulating (PLAG) domain (29-54 amino acids) of rPDPN, have not been developed. Therefore, functional analysis of rPDPN in normal tissues has been limited. Here, we immunized mice with rPDPN peptides (38-51 amino acids) and developed a novel mouse anti-rPDPN MAb, PMab-2 (IgG(1), kappa), which possesses high affinity compared with anti-hPDPN or mPDPN MAbs. The K-D of PMab-2 was determined to be 5.9x10(-10) M. PMab-2 is useful, not only in flow cytometry and Western blot analysis against endogenous rPDPN, which is expressed in rat dermal fibroblast, but also in immunohistochemistry against normal tissues. PMab-2 showed extraordinarily high sensitivity in immunohistochemistry, indicating that PMab-2 is very advantageous for functional analysis of rPDPN.