产生促红细胞生成素的肝细胞癌(Eph)受体是最大的受体酪氨酸激酶家族。EphB4对于胚胎发生过程中的细胞粘附和运动至关重要。病理上,EphB4过表达并导致各种肿瘤的不良预后。因此,应该开发特异性单克隆抗体(mAb)来预测EphB4高表达的多种肿瘤的预后,包括乳腺癌和胃癌.本研究旨在使用基于细胞的免疫和筛选方法开发用于多种应用的特异性抗EphB4mAb。将EphB4过表达的中国仓鼠卵巢(CHO)-K1(CHO/EphB4)细胞免疫入小鼠,我们建立了抗EphB4单克隆抗体(克隆B4Mab-7),适用于流式细胞术,蛋白质印迹,免疫组织化学(IHC)。B4Mab-7与内源性EphB4阳性乳腺癌细胞系反应,MCF-7,但在流式细胞术中不与EphB4敲除的MCF-7(BINDS-52)反应。通过流式细胞术分析CHO/EphB4和MCF-7细胞的解离常数(KD)值确定为2.9×10-9M和1.3×10-9M。分别。B4Mab-7在Westernblot中检测到乳腺癌细胞的EphB4蛋白带,乳腺癌组织免疫组化染色。总之,B4Mab-7对于在各种应用中检测EphB4非常有用。
The erythropoietin-producing hepatocellular carcinoma (Eph) receptors are the largest receptor tyrosine kinase family.
EphB4 is essential for cell adhesion and motility during embryogenesis. Pathologically,
EphB4 is overexpressed and contributes to poor prognosis in various tumors. Therefore, specific monoclonal antibodies (mAbs) should be developed to predict the prognosis for multiple tumors with high EphB4 expression, including breast and gastric cancers. This study aimed to develop specific anti-EphB4 mAbs for multiple applications using the Cell-Based Immunization and Screening method.
EphB4-overexpressed Chinese hamster ovary (CHO)-K1 (CHO/
EphB4) cells were immunized into mice, and we established an anti-EphB4 mAb (clone B4Mab-7), which is applicable for flow cytometry, Western blot, and immunohistochemistry (IHC). B4Mab-7 reacted with endogenous EphB4-positive breast cancer cell line, MCF-7, but did not react with
EphB4-knockout MCF-7 (BINDS-52) in flow cytometry. Dissociation constant (KD) values were determined to be 2.9 × 10-9 M and 1.3 × 10-9 M by flow cytometric analysis for CHO/EphB4 and MCF-7 cells, respectively. B4Mab-7 detected the EphB4 protein bands from breast cancer cells in Western blot, and stained breast cancer tissues in IHC. Altogether, B4Mab-7 is very useful for detecting
EphB4 in various applications.