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Journal of ZheJiang University(Medical Science)  2013, Vol. 42 Issue (5): 511-516    DOI: 10.3785/j.issn.1008-9292.2013.05.006
    
Preparation of anti-B7-H4 monoclonal antibody to investigate B7-H4 expression in pancreatic cancer
HONG Bo1, QIAN Yun2, YAO Hang-Ping3
1.Department of Pathology, the Second Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou 310009, China; 2.Clinical Laboratory, the Second Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou 310009, China; 3.State Key Laboratory for Diagnosis and Treatment of Infectious Diseases, Institute of Infectious Diseases, the First Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou 310003, China;
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Abstract  

Objective: 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.
Methods: 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.
Results: 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 vs 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 vs 3.55±1.12,P<0.01;6.14±0.66 vs 3.70±1.25,P<0.01).The expression level of B7-H4 was not related to patients′ age and gender.
Conclusion: 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.



Key wordsB7-H4      Antigens, CD80      Pancreatic neoplasms      Genes, Neoplasm      Antibodies, Monoclonal      Immunohistochemistry      Lymphatic metastasis      Enzyme-linked immunosorbent assay     
Received: 06 January 2013      Published: 25 September 2013
CLC:  R 74  
Cite this article:

HONG Bo, QIAN Yun, YAO Hang-Ping. Preparation of anti-B7-H4 monoclonal antibody to investigate B7-H4 expression in pancreatic cancer. Journal of ZheJiang University(Medical Science), 2013, 42(5): 511-516.

URL:

https://www.zjujournals.com/med/10.3785/j.issn.1008-9292.2013.05.006     OR     https://www.zjujournals.com/med/Y2013/V42/I5/511


B7-H4在胰腺癌中的表达及其临床意义

目的:制备小鼠抗B7-H4单克隆抗体后研究B7-H4在胰腺癌中的表达情况及临床意义。
方法:以稳定表达B7-H4胞外段的3T3-B7-H4细胞为免疫原,免疫Babl/C小鼠;应用常规杂交瘤技术将免疫的小鼠脾细胞与Sp2/0小鼠骨髓瘤细胞融合,使用间接ELISA方法筛选分泌抗B7-H4 单克隆抗体的杂交瘤细胞株。对所获得的单克隆抗体进行亚型鉴定,使用Western blot、免疫沉淀和免疫组化等方法对单克隆抗体特异性进行鉴定;应用免疫组织化学染色检测B7-H4 分子在胰腺癌中的表达情况,并评价其与临床病理特征的关系。
结果:获得1株亚型为IgM、轻链为κ型的单克隆抗体,经鉴定该单克隆抗体能与B7-H4发生反应。免疫组化显示:胰腺癌组织中B7-H4 在胞浆和(或)胞膜弥漫表达,表达量显著高于正常胰腺组织(P<0.05)。B7-H4在临床肿瘤分级较高患者的胰腺癌组织及有淋巴结转移患者的胰腺癌组织中表达显著增强。
结论:成功获得了特异性抗B7-H4的单克隆抗体,B7-H4 在胰腺癌组织中表达上调,并与患者肿瘤分级和淋巴结转移密切相关。


关键词: B7-H4,  抗原,CD80,  胰腺肿瘤,  基因,肿瘤,  抗体,单克隆,  免疫组织化学,  淋巴转移,  酶联免疫吸附测定 
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