膜结合型抗大鼠FcεRIα单克隆抗体制备及活性鉴定  

Preparation of membrane binding monoclonal antibody anti rat FcεRIα and dectection of its activity

作　　者：李莉[1] 李先兴 徐银海[3] 张玲珍[1] 孔宪涛[1] 仲人前[1] 

机构地区：[1]第二军医大学长征医院实验诊断科,上海200003 [2]长征医院器官移植中心 [3]徐州医学院附属医院检验科,徐州220114

出　　处：《第二军医大学学报》2004年第12期1303-1306,共4页Academic Journal of Second Military Medical University

基　　金：国家自然科学基金(30171200)

摘　　要：目的:建立抗鼠FcεRI单克隆抗体(monoclonal antibody,McAb),为靶向诱导肥大细胞凋亡创造条件。方法:大鼠嗜碱性粒细胞细胞系RBL-2H3免疫的BALB／c小鼠脾细胞与骨髓瘤细胞SP2／0融合,ELISA筛选鼠血清和融合细胞产生的抗体,有限稀释克隆化培养。SDS-PAGE胶电泳、免疫扩散鉴定抗体性质,IgE竞争结合、组胺释放检测抗体生物活性,125I-细胞膜抗体复合物免疫沉淀放射自显影鉴定抗体识别抗原成分。结果:获得2株抗FcεRIα McAb生产细胞株ER-E5.3、ER-C7.4,均为IgG1。培养上清和小鼠腹水获得的抗体效价均大于10-5。流式细胞术分析抗体与RBL-2H3结合率均大于95％,与大鼠胸腺细胞和外周血淋巴细胞(peripheral blood lymphocyte,PBL)无交叉反应,并能竞争性抑制IgE与肥大细胞结合。抗体触发RBL-2H3脱颗粒,组胺释放效应与抗原特异性IgE对肥大细胞的激活效应相同。酶切的Fab片段能与RBL-2H3结合,也能竞争抑制IgE与肥大细胞的结合,但不致RBL-2H3脱颗粒。抗体结合的细胞膜成分为α亚单位。结论:大鼠肥大细胞免疫小鼠与骨髓瘤细胞融合得到2株抗体产生杂交瘤克隆ER-E5.3和ER-C7.4,抗体效价和功能活性达到了实验要求,抗原识别成分为膜FcεRlα。Objective:To induce the mast cell apoptosis by specifically targeting the monoclonal antibody (mAb) against FcεRIα. Methods: Splenocytes of BALB/c mice immunized with rat basophilic leukemia cells line RBL-2H3 were fused with myeloma cells line SP2/0. ELISA assay was used to screen antibody secreting from mouse sera and fused cells. Hybridoma cell lines were cloned by limiting dilution. The character and bioactivity of the antibodies were measured by immune electrophoresis on SDS-PAGE gel,immunodifusion,competitive inhibition and histamine release. The antigen component was verified by analyzing the autoradiography of precipitation of immune complexes of 125I-labeled RBL-2H3 and mAbs. Results: Two monoclonal antibody cell lines ER-E5. 3 and ER-C7. 4 were acquired. The antibodies were specifically against rat FcεRIα and all belonged to IgGl subtype. The titrations of antibodies in supernatant and mouse ascites were higher than 105. Flow cytometry (FCM) showed that the binding rate of antibodies to RBL-2H3 cells was over 95% but no cross-linked activity to rat thymus and peripheral blood lymphocytes (PBL) was detected. The antibodies prevented IgE bind to RBL-2H3 and the histamine release was equivalent to that of DNP-lgE triggered RBL-2H3. Fab prepared by papain-digestion of the whole antibody showed the same activity as whole antibody in self-binding and inhibiting IgE binding to RBL-2H3,but lost the ability to trigger histamine release. Immunoprecipitation and autoradiography demonstrated that the antibody was directly against membrane FcεRIα-subunit. Conclusion: Two hybridoma clones ER-E5. 3 and ER-C7. 4 have been obtained. The titration and activity of the 2 antibodies all meet the requirement for experimental study and the antigen can be specifically recognized by FcεRα.

关 键 词：FcεRIα 抗体 单克隆 肥大细胞 

分 类 号：R392.11[医药卫生—免疫学]