用p[Tj(a-)]外周血淋巴细胞建立抗-PP1P^(k)杂交瘤细胞株

Establishment of human monoclonal hybridoma cell lines specific for PP1P~k antigens from peripheral B lymphocytes of a p[Tj(a-)]donor

目的建立分泌抗-PP1P^(k)(又称抗-Tj^(a))的淋巴母细胞样细胞系(lymphoblastoid cell line,LCL)和单克隆杂交瘤细胞株。方法无菌条件下常规方法制备p[Tj(a-)]血型献血者外周血单个核细胞(Peripheral blood mononuclear cells,PBMC),用EB病毒(Epstein-Barr virus,EBV)转化单个核细胞中的B淋巴细胞,使其增殖形成B淋巴母细胞样细胞克隆。用菠萝酶处理的正常AB型和O型红细胞以及p[Tj(a-)]红细胞筛选培养上清,通过显微吸引分离技术获得分泌特异性抗-PP1P^(k)的LCL,与SHM-D33骨髓瘤细胞进行融合。异源杂交瘤细胞经次黄嘌呤(H)-氨基蝶呤(A)-胸腺嘧啶(T)-脱氧胞嘧啶核苷(D)-毒毛旋花苷(O)即HOTD培养基选择性培养、抗体筛选以及有限稀释亚克隆,建立分泌特异性抗体的单克隆杂交瘤细胞株。结果从O型p[Tj(a-)]献血者外周血B淋巴细胞EBV转化细胞培养上清中,初筛得到与正常红细胞全部凝集,与p[Tj(a-)]红细胞不凝集的单个淋巴母细胞样克隆5E1-E5,初步鉴定培养上清具有抗-PP1P^(k)特异性。随后利用杂交瘤技术,将5E1-E5细胞与SHM-D33骨髓瘤细胞进行融合,建立了持续稳定分泌IgM抗-PP1P^(k)的单克隆杂交瘤细胞株。结论本研究通过EBV转化和杂交瘤技术建立了分泌抗-PP1P^(k)的人单克隆细胞株,这项工作使大规模筛选稀有p[Tj(a-)]血型成为可能,对我国稀有血型库的建设具有重要意义。

Objective To establish lymphoblastoid cell line(LCL)and human monoclonal antibody cell lines against PP1P^(k) antigens from a donor with rare p[Tj(a-)]phenotype.Methods Peripheral blood mononuclear cells(PBMCs)of an O type p[Tj(a-)]donor were prepared and transformed with Epstein-Barr virus(EBV)in vitro.Normal AB and O type red blood cells treated with bromelain as well as p[Tj(a-)]were used to screen the culture supernatant.The transformed LCL secreting antibodies to PP1P^(k) antigens was picked up and then hybridized with the SHM-D33 cell line using electric fusion technique.The hybridomas were performed by HOTD selective culture,antibody screening and limited dilution subcloning.Results We established a lymphoblastoid cell line 5E1-E5 and several monoclonal hybridoms cell lines secreting anti-PP1P^(k) immunoglobulin M from EBV transformed B lymphocytes of a p[Tj(a-)]type donor with anti-PP1P^(k) successfully.Conclusion Our work made it possible for screening rare p[Tj(a-)]blood type in donor population in China using human monoclonal anti-PP1P^(k) antibodies,which was important for the establishment of rare blood group banks in China.