抗人蛋白酶激活受体3单克隆抗体的制备与鉴定

Preparation and identification of monoclonal antibodies against human proteinase-activated receptor-3

目的:制备并鉴定抗人蛋白酶激活受体3(PAR3)单克隆抗体(mAb)。方法:以人PAR3为免疫原免疫BALB/c小鼠,运用杂交瘤技术制备抗PAR3 mAb。采用捕获ELISA法鉴定mAb的Ig亚类;通过ELISA、流式细胞分析、激光共聚焦显微镜技术、免疫组织化学染色法、斑点杂交技术鉴定mAb的特异性。结果:获得2株可稳定分泌抗人PAR3 mAb的杂交瘤细胞,其分泌的mAb均为IgM。斑点杂交技术分析表明,其mAb能与PAR3抗原有效结合。免疫组织化学染色表明,mAb与肺组织中的巨噬细胞、结肠组织中的淋巴细胞及疑似肥大细胞、扁桃体和包皮组织中的淋巴细胞的反应呈阳性。流式细胞仪分析及激光共聚焦显微镜观察显示,2株mAb与人肺腺癌A549细胞胞内及膜上的PAR3均呈阳性反应。结论:成功地制备抗PAR3 mAb,为变态反应性和炎症性疾病的研究提供了有用的试剂。

Aim： To prepare and identify monoclonal antibodies （mAbs） against human proteinase-activated receptor- 3 （hPAR3）. Methods：BALB/c mice were immunized with hPAR3, and PAR3 mAb was prepared by hybridoma techonology. The specificity of mAbs was characterized by enzyme-linked immunosorbent assay （ELISA） ,The speaficity of mAb was characterized by flow cytometry （FCM） , confocal laser scanning microscopy （CLSM） , immunohistochemical staining and Dot blot. Results： 2 hybridoma cells that secreted the mAbs to PAR3 were obtained. 2 mAbs were IgM. Dot blot analysis showed that the mAbs could combine with PAR3 antigen efficiently. Immunohistochemical staining showed that the reactivities of the 2 mAbs to the macrophage cells in lung, mast cell-like cells in colon, lymphocyte cells in tonsil and prepuce tissues were positive. The result of FCM and CLSM showed that the 2 mAbs recognized PAR3 expressed on the A549 cells. Conclusion： The mAbs against hPAR3 were prepared successfully, which provides valuable reagent for studies on allergic and inflammatory diseases.