hK6单克隆抗体制备及在胃和乳房肿瘤组织中的初步应用  

Preparation of Monoclonal Antibody Against hK6 and Primary Application in Diagnosis on the Breast Cancer Tissues

作　　者：张华[1] 胡成进[1] 王燕[1] 

机构地区：[1]济南军区总医院实验诊断科,250031

出　　处：《放射免疫学杂志》2013年第5期607-610,共4页Journal of Radioimmanology

基　　金：全军"十一五"面上项目;编号:06MA101

摘　　要：目的:原核表达人组织激肽释放酶6(human kallikrein 6,hK6),采用杂交瘤技术制备其单克隆抗体,建立免疫组织化学方法,检测hK6在胃癌、乳腺癌组织中的表达情况。方法:通过原核表达hK6融合蛋白免疫BALB/c小鼠,采用杂交瘤技术制备特异性单克隆抗体,并用间接ELISA、SDS-PAGE及Western-blot鉴定抗体,protein A agarose亲和层析柱纯化抗体,并以此抗体建立免疫组织化学方法检测人胃癌、乳腺癌组织中hK6表达情况。结果:成功筛选出4株高分泌型特异性杂交瘤细胞株,其制备抗体可与hK6融合蛋白发生特异性结合;免疫组织学显示,hK6主要分布在胃癌细胞胞浆中,成阳性表达,而在癌旁胃黏膜中成阴性表达;在乳腺癌组织中定位于胞浆,成弱阳性表达,癌旁组织成阴性表达。结论:成功制备了高纯度、特异性的抗hK6单克隆抗体;该抗体为深入研究hK6功能和建立高特异性、高敏感性检测组织和体液中hK6的方法奠定基础。Objeotive To preparate monoclonal antibody against hK6 and using hybridoma technique to prepare monoclonal anti-body and establish immunohistochemical method to investigate the distribution of hK6 in gastric and breast cancer tissues. Methods A BALB/c mice was immunized with the hK6 fusion protein to preparate McAbs against hK6. And it was identified by indirect ELISA assay and Western-blot and purified by protein A agarose. Immunocytochemical staining was applied for hK6 expression in gaster and breast cancer tissues. Results 4 hybridoma cell strains were selected. And the McAbs against hK6 were specific for hK6 fusion pro- tein and reacted with a 25000 dalton molecule, hK6 was mainly localized in cytoplasm. The positive reaction was found in tissues of gastric cancer or breast cancer or normal ovary by immunohistochemical staining. Conclusion This article successfully prepared the McAbs against hK6 which showed high titer, good specificity and high sensitivity. It provided the basis for further research into func- tions of hK6 and into detecting hK6 in human tissue and fluid.

关 键 词：人组织激肽释放酶6 单克隆抗体 免疫组织化学 

分 类 号：R392-33[医药卫生—免疫学]