An optimized electrofusion-based protocol for generating virus-specific human monoclonal antibodies
An optimized electrofusion-based protocol for generating virus-specific human monoclonal antibodies
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DOI：
10.1016/j.jim.2008.04.008
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发表时间：
2008-07-31
影响因子：
2.2
通讯作者：
Crowe, James E., Jr.
中科院分区：
文献类型：
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作者：
Yu, Xiaocong;McGraw, Patricia A.;Crowe, James E., Jr.
我们试图开发和优化一种基于杂交瘤的技术，以产生分泌病毒特异性单抗的人类杂交瘤，用于临床诊断和治疗。我们开发了一种新的电融合方案，用于将EB病毒(EBV)转化的人B细胞与骨髓瘤伙伴有效融合。我们测试了7个骨髓瘤细胞系，当使用HMMA 2.5系时，获得了最高的效率。我们通过改进电融合前后的细胞处理以及改变细胞比例、融合介质和其他实验参数来优化电融合过程。我们的融合效率显著提高到0.43%，比以前基于聚乙二醇基或其他电融合方法的效率有了显著的提高。使用优化的方案，我们获得了完全分泌针对两种主要人类呼吸道病原体的人类单抗的人杂交瘤细胞株：呼吸道合胞病毒(RSV)和流感H3N2疫苗病毒株。总之，我们开发了一种有效和常规的方法来产生分泌功能性人类病毒特异性单抗的人类杂交瘤。(C)2008爱思唯尔B.V.保留所有权利。
We sought to develop and optimize a hybridoma-based technology for generating human hybridomas that secrete virus-specific monoclonal antibodies for clinical diagnosis and therapy. We developed a novel electrofusion protocol for efficiently fusing Epstein-Barr virus (EBV)-transformed human B cells with myeloma partners. We tested seven myeloma cell lines and achieved highest efficiency when the HMMA 2.5 line was used. We optimized the electrofusion process by improving cell treatments before and after electrofusion as well as varying cell ratios, fusion medium and other experimental parameters. Our fusion efficiency increased remarkably to 0.43%, a significant improvement over the efficiency of previous PEG-based or other electrofusion methods. Using the optimized protocol, we obtained human hybridomas that secrete fully human monoclonal antibodies against two major human respiratory pathogens: respiratory syncytial virus (RSV) and an influenza H3N2 vaccine virus strain. In conclusion, we have developed an efficient and routine approach for the generation of human hybridomas secreting functional human virus-specific monoclonal antibodies. (C) 2008 Elsevier B.V. All rights reserved.