利用D--外周血B细胞构建单克隆抗-C杂交瘤细胞株  

Preparation of human monoclonal anti-C cell line from peripheral blood B lymphocytes of D--donor

作　　者：郭忠慧[1] 赵俸涌[1] 张德梅 向东[1] 张嘉敏[1] 杨颖[1] 李勤[1] 杨启修[1] 王晨[1] 朱自严[1] GUO Zhonghui;ZHAO Fengyong;ZHANG Demei;XIANG Dong;ZHANG Jiamin;YANG Ying;LI Qin;YANG Qixiu;WANG Chen;ZHU Ziyan(Shanghai Blood Center,Shanghai 200051,China;Taiyuan Blood Center)

机构地区：[1]上海市血液中心,上海200051 [2]太原市血液中心

出　　处：《中国输血杂志》2022年第4期400-404,共5页Chinese Journal of Blood Transfusion

基　　金：上海市卫生健康委员会科研课题面上项目(202040501)。

摘　　要：目的以1例稀有的O型D--献血者外周血单个核细胞为实验对象,制备分泌Rh血型系统单克隆抗体的杂交瘤细胞株。方法无菌分离外周血白细胞,经淋巴细胞分离液分离得到单个核细胞(PBMCs),利用B95-8培养上清中的EB病毒(EBV),转化PBMCs中的B淋巴细胞使其母细胞化。血清学筛选分泌特异性抗体的单一B细胞克隆并扩大培养,与SHM-D33瘤细胞在电刺激下融合,经含有次黄嘌呤(Hypoxantine,H)、氨基蝶呤(Aminopterin,A)、胸腺嘧啶核苷(Thymidine,T)、脱氧胞嘧啶核苷(2-Deoxycytide,D)、毒毛旋花苷(Ouabain,O)的培养基,即HOTD(HATD+Ouabain)选择性培养、微量板抗体筛选以及有限稀释亚克隆,建立分泌特异性抗体的杂交瘤细胞株,血清学和流式细胞试验鉴定抗体特异性。结果从D--外周血转化B淋巴细胞培养上清中,初筛得到与R;R;(DCe/DCe)O型红细胞凝集,与R;R;(DcE/DcE)O型红细胞不凝集的单个细胞克隆3A6-C6,初步鉴定抗体特异性为抗-C,与SHM-D33骨髓瘤细胞杂交后建立了持续稳定分泌IgM抗-C的杂交瘤细胞株。结论本研究从外周血B细胞开始构建单克隆杂交瘤细胞株,为Rh血型系统血清学单克隆试剂的研发和改造奠定了基础。Objective To establish human hybridoma cell lines,secreting monoclonal antibody against antigens of Rh blood system,from a donor with rare D--phenotype.Methods Peripheral blood B lymphocytes of an O type female donor,lacking C/c/E/e antigens on her erythrocyte,were transformed with Epstein-Barr virus(EBVs).EBVs were harvested from the cultural supernatant of B95-8 cells.The transformed lymphoblastoid cell line(LCL)secreting antibodies to C antigens were picked up and then hybridized with the myeloma SHM-D33 using electric fusion technique.Hybridoma cells were selected by HATD-Ouabain(HOTD)(Hypoxantine,Aminopterin,Thymidine,2-Deoxycytide,and Ouabain)culture medium,microplate antibody screening and limited dilution subcloning.The monoclonal antibody was assayed by serological test and was confirmed by flow cytometry(FCM).Results From the cultural supernatant of D--peripheral blood transformed B lymphocytes,3 A6-C6,which agglutinated with R;R;(DCe/DCe)O-type RBCs but not with R;R;(DcE/DcE)O-type RBCs,was screened and preliminarily identified as anti-C.We established a hybridoma cell line secreting anti-C immunoglobulin M from B cells of D--individual successfully after hybridization with SHM-D33 myeloma cells.Conclusion The study had laid the groundwork for future research and development of human monoclonal antibodies against Rh antigens of RBC in future for diagnosis and screening purpose.

关 键 词：D--表型 人单克隆抗体 抗-C 杂交瘤技术 

分 类 号：R457.1[医药卫生—治疗学]