[1]王顺兰)△,何韶衡)#.抗人蛋白酶激活受体4单克隆抗体的制备与鉴定[J].郑州大学学报(医学版),2009,(06):1188-1190.
 WANG Shunlan),HE Shaoheng).Preparation and characterization of monoclonal antibody against proteaseactivated receptor 4[J].JOURNAL OF ZHENGZHOU UNIVERSITY(MEDICAL SCIENCES),2009,(06):1188-1190.
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抗人蛋白酶激活受体4单克隆抗体的制备与鉴定 ()
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《郑州大学学报(医学版)》[ISSN:1671-6825/CN:41-1340/R]

卷:
期数:
2009年06期
页码:
1188-1190
栏目:
论著
出版日期:
2009-11-01

文章信息/Info

Title:
Preparation and characterization of monoclonal antibody against proteaseactivated receptor 4
作者:
王顺兰1)何韶衡2)#
1)海口市人民医院中心实验室 海口 5207082)南京医科大学附属第一医院中心实验室 南京 210029
Author(s):
WANG Shunlan1)HE Shaoheng2)
1)Center Laboratory,the People’s Hospital of Haikou,Haikou 5702082)Center Laboratory,the First Affiliated Hospital,Nanjing Medical University, Nanjing 210029
关键词:
蛋白酶激活受体4单克隆抗体杂交瘤细胞A549细胞
Keywords:
proteaseactivated receptor 4monoclonal antibodyhybridoma cellA549 cell
分类号:
R392.11
摘要:
制备抗人蛋白酶激活受体4(PAR4)单克隆抗体(mAb)。方法:以hPAR4为免疫原免疫BALB/c小鼠,运用杂交瘤技术制备抗PAR4 mAb。采用Ig类与亚类鉴定试剂盒鉴定mAb的Ig类型,采用ELISA、点杂交、免疫组化染色法(人结肠、扁桃体及包皮组织)、流式细胞术、激光共聚焦显微镜(CLSM)技术鉴定mAb的特异性。结果:获得3株可稳定分泌抗PAR4 mAb的杂交瘤细胞,其分泌的mAb均为IgM。免疫组化染色表明,mAb将人结肠组织中的腺上皮细胞、疑似肥大细胞和淋巴细胞,扁桃体组织中的淋巴细胞和包皮组织中的肥大细胞染成褐色。流式细胞术分析显示,3株mAb与A549细胞的PAR4均呈阳性反应。CLSM观察发现,荧光标记的阳性反应物主要位于A549细胞的胞质和胞膜上。结论:成功制备了抗PAR4 mAb,为进一步的研究工作提供了有用的试剂。
Abstract:
To prepare and identify the monoclonal antibody (mAb) against proteaseactivated receptor 4 (PAR4).Methods:BLAB/c mice were immunized with specific PAR4 polypeptide by multiplesite hypodermic injection. The spleen cells of immunized mice were fused with NS1 myeloma cells. Indirect ELISA was used to screen the hybridoma culture supernants. The specificity of mAb were characterized by ELISA, Dot blot, immunohistochemistry, flow cytometry,and confocal laser scanning microscopy (CLSM).Results: Three hybridoma cell lines stably screting mAb against PAR4 were successfully obtained, these mAbs were IgM isotype,and the titer of antibody in supernants were 13,110,and 110, respectively. Immunohistochemical staining revealed that the reactivity of mAb to epithelial cells, mastlike cells and lymphocytes in colon; lymphocytes in tonsil and mast cells in foreskin.The result of flow cytometry showed that the mAb recognized PAR4 of A549 cells. CLSM examination confirmed that the fluorescent markers were localized in both cytomembrane and cytoplasm of A549 cells.Conclusion: The mAb against PAR4 is useful for investigate hematologic disease, allergic disorder and inflammation that are related with PAR4.

参考文献/References:

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[2]康振华,王为忠,陈冬利,等. 抗人RANTES分子单克隆抗体的制备及特性鉴定[J].细胞与分子免疫学杂志,2005,21(3):322
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备注/Memo

备注/Memo:
△女,1981年生,硕士,研究方向:变态反应性疾病的发病机制,Email:shunlnawang@163.com #通讯作者,男,1959年生,博士,教授,研究方向:变态反应性疾病,Email:shoahenghe@hotmail.com
更新日期/Last Update: 2010-05-14