Hong Bo, Qian Yun, Yao Hang-Ping
Department of Pathology, the Second Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou 310009, China.
Zhejiang Da Xue Xue Bao Yi Xue Ban. 2013 Sep;42(5):511-6.
To prepare a monoclonal antibody (mAb) against extracellular domain of B7-H4 and to investigate the expression of B7-H4 in pancreatic cancer tissue with the prepared mAb.
Balb/c mice were immunized with 3T3-B7-H4 cells and the splenic cells of the immunized mice were fused with Sp2/0 myeloma cells by conventional hybridoma techniques. An indirect ELISA method using 3T3-B7-H4 lysate as antigen was established to screen antibody-producing hybridoma cell lines. Western blott, immunoprecipitation (IP), and immunohistochemistry (IHC) were applied to characterize the mAb. Immunohistochemical staining was used to detect the expression of B7-H4 in human pancreatic cancer tissue. The correlation of B3-H4 expressions and pathological features of pancreatic cancer was analyzed.
A hybridoma cell line secreting mAb against B7-H4 was obtained. The subclass of this mAb was IgM, and the light chain was Kappa. Western blot and IP showed that the mAb specifically recognized B7-H4. IHC staining revealed that the mAb stained in a predominantly diffuse plasmalemmal or cytoplasmic pattern when applied to certain tumor tissues. The B7-H4 was diffusely expressed in the cytoplasma and/or membrane of pancreatic cancer tissue, which was much higher than that expressed in normal pancreatic tissue (4.00 ± 1.44 compared with 1.12 ± 0.78, P ± 0.01). The expression of B7-H4 was higher in pancreatic cancer tissues with higher pathological grade or with lymph node metastasis as compared with that in pancreatic cancer tissues with lower grade or with no lymph mode metastasis (6.10 ± 0.72 compared with 3.55 ± 1.12,P<0.01: 6.14 ± 0.66 compared with 3.70 ± 1.25,P<0.01). The expression level of B7-H4 was not related to patients'age and gender.
Monoclonal antibody against B7-H4 with high activity and specificity has been prepared successfully. The expression of B7-H4 in pancreatic cancer tissue is up-regulated,which is closely related to the tumor grade and lymph node metastasis in pancreatic cancer.
制备抗B7-H4胞外区的单克隆抗体，并利用所制备的单克隆抗体研究胰腺癌组织中B7-H4的表达情况。
用3T3-B7-H4细胞免疫Balb/c小鼠，采用常规杂交瘤技术将免疫小鼠的脾细胞与Sp2/0骨髓瘤细胞进行融合。建立以3T3-B7-H4裂解液为抗原的间接ELISA方法筛选分泌抗体的杂交瘤细胞株。应用蛋白质免疫印迹法（Western blott）、免疫沉淀法（IP）和免疫组织化学法（IHC）对单克隆抗体进行鉴定。采用免疫组织化学染色法检测人胰腺癌组织中B7-H4的表达情况，并分析B7-H4表达与胰腺癌病理特征的相关性。
获得一株分泌抗B7-H4单克隆抗体的杂交瘤细胞株。该单克隆抗体的亚类为IgM，轻链为κ链。蛋白质免疫印迹法和免疫沉淀法显示该单克隆抗体能特异性识别B7-H4。免疫组织化学染色显示，将该单克隆抗体应用于某些肿瘤组织时，主要呈弥漫性细胞膜或细胞质染色模式。B7-H4在胰腺癌组织的细胞质和/或细胞膜中呈弥漫性表达，其表达水平远高于正常胰腺组织（分别为4.00±1.44和1.12±0.78，P<0.01）。与低病理分级或无淋巴结转移的胰腺癌组织相比，高病理分级或有淋巴结转移的胰腺癌组织中B7-H4的表达更高（分别为6.10±0.72和3.55±1.12，P<0.01；6.14±0.66和3.70±1.25，P<0.01）。B7-H4的表达水平与患者的年龄和性别无关。
成功制备了具有高活性和特异性的抗B7-H4单克隆抗体。胰腺癌组织中B7-H4的表达上调，且与胰腺癌的肿瘤分级和淋巴结转移密切相关。
