【摘要】 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。更多还原

【Abstract】 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.更多还原