Kwekkeboom J, van Oosten M, de Boer M, van Alphen L, Mevissen M L, Lindhout E, Tager J M, de Groot C
Laboratory of Cell Biology and Histology, University of Amsterdam, Academic Medical Center, Netherlands.
J Immunol Methods. 1993 Mar 15;160(1):117-27. doi: 10.1016/0022-1759(93)90015-y.
A new, efficient procedure for the generation of human monoclonal antibodies has been developed. The procedure is based on the activation of human B cells in microwells by murine thymoma EL4B5 cells. This mode of B cell stimulation leads to proliferation of at least one per eight of human B cells and to a high rate of antibody production. Subsequently, supernatants of the microwells are screened by ELISA for the presence of antibody of the desired specificity and B cells from selected wells are hybridized by electroporation. To optimize the procedure, the kinetics of the B cell expansion induced by EL4B5 cells were analysed. Counting and phenotyping of cultured cells at different time points indicated that the peak of B cell expansion occurred at day 5 for tonsil B cells (16-fold increase) and at day 7 for peripheral blood B cells (20-fold increase). The B cells did not merely proliferate but also differentiated, as indicated by loss of CD20 expression and increase of CD38 expression. At the peak of B cell expansion, B cells could be hybridized efficiently with myeloma cells. The majority of the resultant hybridomas secreted human immunoglobulin. The efficiency of the procedure is exemplified by the generation of hybridomas secreting human IgG against Haemophilus influenzae from limited numbers of either human tonsil B lymphocytes or peripheral blood B lymphocytes.
已开发出一种用于生成人单克隆抗体的新型高效方法。该方法基于鼠胸腺瘤EL4B5细胞在微孔中激活人B细胞。这种B细胞刺激模式导致每八个B细胞中至少有一个增殖，并产生高比率的抗体。随后，通过ELISA筛选微孔上清液中所需特异性抗体的存在，并通过电穿孔将所选孔中的B细胞进行杂交。为了优化该方法，分析了EL4B5细胞诱导的B细胞扩增动力学。在不同时间点对培养细胞进行计数和表型分析表明，扁桃体B细胞在第5天达到B细胞扩增峰值（增加16倍），外周血B细胞在第7天达到峰值（增加20倍）。B细胞不仅增殖，而且分化，这表现为CD20表达丧失和CD38表达增加。在B细胞扩增峰值时，B细胞可与骨髓瘤细胞有效杂交。大多数所得杂交瘤分泌人免疫球蛋白。从有限数量的人扁桃体B淋巴细胞或外周血B淋巴细胞中生成分泌抗流感嗜血杆菌人IgG的杂交瘤，例证了该方法的效率。
