抗蛋白酶激活受体-2单克隆抗体的制备与鉴定

发布时间：2018-11-16 16:08

【摘要】：目的:制备出一种具有多种用途的抗蛋白酶激活受体-2(PAR-2)单克隆抗体(mAb)。方法:用人工合成的11肽PAR-2作为免疫原,采用皮下多点注射方法免疫BALB/c小鼠,取其脾细胞与NS-1骨髓瘤细胞融合,杂交瘤细胞采用间接ELISA法筛选。通过ELISA、Dot blot、免疫组化染色、流式细胞分析、激光共聚焦显微镜技术鉴定mAb的特异性。结果:获得1株可稳定分泌抗PAR-2 mAb的杂交瘤细胞。其分泌的mAb为IgM型,ELISA法检测杂交瘤细胞培养上清效价为1:16;点杂交分析表明此抗体可特异性结合PAR-2;免疫组化染色显示该单克隆抗体与人肺组织中的肺泡上皮细胞和平滑肌细胞,结肠组织中的腺上皮细胞和疑似淋巴细胞,扁桃体中的淋巴细胞,包皮组织中的鳞状上皮细胞呈阳性反应;流式细胞仪分析显示与人肺腺癌细胞系A549细胞中的PAR-2呈阳性反应;激光共聚焦扫描显微镜观察发现荧光标记的阳性反应物位于A549细胞的胞膜和胞浆。结论:成功地制备出可用于免疫组化和点杂交的抗PAR-2单克隆抗体,为PAR-2相关性疾病的研究提供了有用的工具。
[Abstract]:Objective: to prepare a multi-purpose monoclonal antibody (mAb).) against protease activated receptor-2 (PAR-2). Methods: BALB/c mice were immunized with synthetic 11-peptide PAR-2. Spleen cells were fused with NS-1 myeloma cells and hybridoma cells were screened by indirect ELISA method. The specificity of mAb was identified by ELISA,Dot blot, immunohistochemical staining, flow cytometry and laser confocal microscopy. Results: a hybridoma cell which could secrete anti PAR-2 mAb stably was obtained. The mAb secreted was IgM type. The titer of the supernatant of hybridoma cell culture detected by ELISA assay was 1: 16. Dot blot analysis showed that the antibody could specifically bind to PAR-2;. Immunohistochemical staining showed that the monoclonal antibody was associated with alveolar epithelial cells and smooth muscle cells in human lung tissue, glandular epithelial cells and suspected lymphocytes in colon tissue, lymphocytes in tonsils, The squamous epithelial cells in the prepuce tissue showed positive reaction. Flow cytometry analysis showed that PAR-2 was positive in human lung adenocarcinoma cell line A549, and the fluorescent labeled positive reaction was located in the membrane and cytoplasm of A549 cell by confocal laser scanning microscopy. Conclusion: monoclonal antibodies against PAR-2 can be successfully prepared for immunohistochemistry and dot hybridization, which provide a useful tool for the study of PAR-2 related diseases.
【作者单位】：汕头大学医学院变态反应学与炎症学研究所汕头大学医学院变态反应学与炎症学研究所汕头大学医学院变态反应学与炎症学研究所汕头大学医学院变态反应学与炎症学研究所汕头大学医学院变态反应学与炎症学研究所
【基金】：广东省重点科技计划资助项目(2003831502) 广东省自然科学基金(04106122)
【分类号】：R392.11

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