摘要
目的制备抗人分泌性白细胞蛋白酶抑制剂(hSL-PI)单克隆抗体(mAb)。方法以hSLPI为免疫BALB/c小鼠,运用杂交瘤技术制备抗hSLPI mAb。采用Ig类与亚类鉴定试剂盒鉴定mAb的Ig亚类;通过ELISA、Western blot、免疫组化染色法、流式细胞术、激光共聚焦显微镜技术鉴定mAb的特异性。结果获得4株可稳定分泌抗hSLPI mAb的杂交瘤细胞,其分泌的mAb均为IgM。Western blot分析表明,此株mAb所识别的靶分子的相对分子质量(Mr)约为12000。免疫组化染色表明,mAb与肺和结肠组织的上皮细胞、疑似肥大细胞、扁桃体和包皮组织的疑似肥大细胞的反应性均呈阳性。流式细胞术分析显示,4株mAb与人肺腺癌细胞系A549细胞中的SLPI均呈阳性反应。激光共聚焦扫描显微镜观察,荧光标记的阳性反应物主要位于A549细胞的胞质内。结论成功地制备抗hSLPI mAb,为变态反应性疾病和炎症性疾病的研究工作提供了有用的试剂。
AIM： To prepare and identify monoclonal antibodies （mAbs） against human secretory leukocyte protease inhibitor （hSLPI）. METHODS： BALB/c mice were immunized with hSLPI, and hybridoma cell lines were obtained by fusing mouse spleen cells with myeloma NS-1 cells. The specificity of mAbs were characterized by ELISA, Western blot, immunohistochemical staining, flow cytometry （FCM） and confocal laser scanning microscopy （CLSM）. RESULTS： Four hybridoma cells which secreted the mAbs to hSLPI were obtained. 4 mAbs were IgM. Western blot analysis showed that the mAbs could recognize a target molecule with relative molecular mass of 12 000. Immunohistochemical staining revealed that the reactivities of 4 mAbs to the epithelial cells in lung and colon tissues, mast cell-like cells in lung, colon, tonsil and prepuce tissues were positive. The result of FCM showed that the 4 mAbs recognized SLPI expressed in A.549 cells. CLSM examination confirmed that the fluorescent markers were mainly localized in the cytoplasm of A549 cells. CONCLUSION： The mAbs against hSLPI are prepared successfully, which provides valuable tool for studies on allergic and inflammatory diseases.
出处
《细胞与分子免疫学杂志》
CAS
CSCD
北大核心
2006年第1期54-57,共4页
Chinese Journal of Cellular and Molecular Immunology
基金
国家自然科学基金资助项目(No.30471601)
广东省科技计划重点项目(No.2003B31502)
广东省自然科学基金重点项目(No.04106122)
李嘉诚先生捐资款专项经费(No.C0200001)
关键词
人分泌性白细胞蛋白酶抑制剂
单克隆抗体
特性鉴定
human secretory leukocyte protease inhibitor
monoclonal antibodies
characterization
