Characterization of a Novel Anti-Human HB-EGF Monoclonal Antibody Applicable for Paraffin-Embedded Tissues and Diagnosis of HB-EGF-Related Cancers.
Characterization of a Novel Anti-Human HB-EGF Monoclonal Antibody Applicable for Paraffin-Embedded Tissues and Diagnosis of HB-EGF-Related Cancers.
复制标题
DOI：
10.1089/mab.2015.0062
复制
发表时间：
2016-04
影响因子：
--
通讯作者：
Mekada E
中科院分区：
文献类型：
--
作者：
Iwamoto R;Takagi M;Akatsuka J;Ono K;Kishi Y;Mekada E
肝素结合EGF样生长因子（HB-EGF）是EGF生长因子家族的一员，可结合并激活EGF受体（EGFR/ErbB1）和ErbB4。HB-EGF在包括癌症在内的病理生理过程中起着关键作用。因此，用于HB-EGF检测的单克隆抗体（mab）可能成为HB-EGF相关癌症和其他疾病的治疗性诊断的重要工具。然而，迄今为止，很少有单克隆抗体，特别是那些适用于免疫组织化学（IHC）的单克隆抗体被建立。在这项研究中，我们用人细胞表达的重组人HB-EGF蛋白免疫小鼠，获得了杂交瘤来源的mAb 2-108克隆。mAb 2-108特异性结合人HB-EGF，但不与小鼠HB-EGF结合，即使在还原条件下，对未固定和多聚甲醛固定的细胞也能成功进行免疫印迹、免疫沉淀和免疫荧光。值得注意的是，该单抗在石蜡包埋肿瘤标本的免疫组化中有效。表位定位分析显示mAb 2-108识别HB-EGF的n端原结构域。这些结果表明，这种新的抗HB-EGF mAb 2-108将有助于HB-EGF相关癌症的诊断，并将成为HB-EGF基础和临床研究的有力工具。
Heparin-binding EGF-like growth factor (HB-EGF) is a member of the EGF family of growth factors that bind to and activate the EGF receptor (EGFR/ErbB1) and ErbB4. HB-EGF plays pivotal roles in pathophysiological processes, including cancer. Thus, monoclonal antibodies (mAbs) for HB-EGF detection could be an important tool in the therapeutic diagnosis of HB-EGF-related cancers and other diseases. However, few mAbs, especially those applicable for immunohistochemistry (IHC), have been established to date. In this study, we generated a clone of hybridoma-derived mAb 2-108 by immunizing mice with recombinant human HB-EGF protein expressed by human cells. The mAb 2-108 specifically bound to human HB-EGF but not to mouse HB-EGF and was successful in immunoblotting, even under reducing conditions, immunoprecipitation, and immunofluorescence for unfixed as well as paraformaldehyde-fixed cells. Notably, this mAb was effective in IHC of paraffin-embedded tumor specimens. Epitope mapping analysis showed that mAb 2-108 recognized the N-terminal prodomain in HB-EGF. These results indicate that this new anti-HB-EGF mAb 2-108 would be useful in the diagnosis of HB-EGF-related cancers and would be a strong tool in both basic and clinical research on HB-EGF.