[抗人分泌型白细胞蛋白酶抑制剂单克隆抗体的制备与鉴定]

[Preparation and characterization of monoclonal antibodies against human secretory leukocyte protease inhibitor].

作者信息

Chen Yu-zhen, He Shao-heng, Zhou Yan-chun, Huang Tian, Liu Yue-ning, Chen Lin-fei

机构信息

Research Institute of Allergy and Inflammation, Shantou University Medical College, Shantou 515031, China.

出版信息

Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi. 2006 Jan;22(1):54-7.

PMID:16388746

Abstract

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 A549 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.

摘要

目的

制备并鉴定抗人分泌型白细胞蛋白酶抑制因子（hSLPI）的单克隆抗体（mAbs）。

方法

用hSLPI免疫BALB/c小鼠，通过将小鼠脾细胞与骨髓瘤NS-1细胞融合获得杂交瘤细胞系。采用酶联免疫吸附测定（ELISA）、蛋白质印迹法（Western blot）、免疫组织化学染色、流式细胞术（FCM）和共聚焦激光扫描显微镜技术（CLSM）对单克隆抗体的特异性进行鉴定。

结果

获得了4株分泌抗hSLPI单克隆抗体的杂交瘤细胞。4种单克隆抗体均为IgM。蛋白质印迹分析表明，这些单克隆抗体能够识别相对分子质量为12 000的靶分子。免疫组织化学染色显示，4种单克隆抗体对肺和结肠组织中的上皮细胞、肺、结肠、扁桃体和包皮组织中的类肥大细胞呈阳性反应。流式细胞术结果显示，这4种单克隆抗体可识别A549细胞中表达的SLPI。共聚焦激光扫描显微镜检查证实，荧光标记主要定位于A549细胞的细胞质中。