Shirahata S, Katakura Y, Teruya K
Laboratory of Cellular Regulation Technology, Graduate School of Genetic Resources Technology, Kyushu University, Fukuoka, Japan.
Methods Cell Biol. 1998;57:111-45. doi: 10.1016/s0091-679x(08)61575-7.
Cell hybridization is one of the most basic cytotechnologies. The hemagglutinating virus of Japan was first used to cause cell fusion; however, polyethylene glycol is widely used now because of simplicity of procedure. This chapter first explains the principles of cell hybridization methods and then describes the practical protocols for preparing mouse hybridomas using polyethylene glycol. So far, lack of an excellent human fusion partner cell line that has high fusion efficiencies and does not produce immunoglobulin has hindered the spread of human-human hybridoma preparation methods. In the authors' laboratory NAT-30 and HO-323, human parent cell lines with high fusion efficiencies, have been established to prepare many hybridoma cell lines producing cancer-specific human monoclonal antibodies. Because NAT-30 and HO-323 cell lines are IgM producers, it is difficult to obtain IgG-producing hybridomas because the types of immunoglobulin produced by hybridomas are strongly affected by the characteristics of parent cells. Thus a nonimmunoglobulin-producing human parent cell line, A4H12, derived from human T lymphoma was established that can efficiently obtain IgG-producing human hybridomas. Another problem with preparing human hybridomas is that it is difficult to obtain B lymphocytes immunized with optional antigens for ethical reasons. To overcome this problem, in vitro immunization methods have been developed that allow exposure of a large number of B lymphocytes to cultured cancer cell or soluble antigens. The section on human hybridomas explains human fusion partners, in vitro immunization methods, and the preparation of human-human hybridomas using an electrofusion method. Finally, the application of human monoclonal antibodies to medical uses and the preparation of supranatural monoclonal antibodies are reviewed. These include multifunctional monoclonal antibodies and altered monoclonal antibodies having increased affinity and specificity by exchanging or modifying light chains.
细胞杂交是最基本的细胞技术之一。日本血凝病毒最初被用于诱导细胞融合；然而，由于操作简便，聚乙二醇现在被广泛使用。本章首先解释细胞杂交方法的原理，然后描述使用聚乙二醇制备小鼠杂交瘤的实际方案。到目前为止，缺乏具有高融合效率且不产生免疫球蛋白的优良人类融合亲本细胞系，阻碍了人-人杂交瘤制备方法的推广。在作者的实验室中，已建立了具有高融合效率的人类亲本细胞系NAT-30和HO-323，用于制备许多产生癌症特异性人类单克隆抗体的杂交瘤细胞系。由于NAT-30和HO-323细胞系产生IgM，因此难以获得产生IgG的杂交瘤，因为杂交瘤产生的免疫球蛋白类型受亲本细胞特性的强烈影响。因此，建立了一种源自人T淋巴瘤的不产生免疫球蛋白的人类亲本细胞系A4H12，它可以有效地获得产生IgG的人类杂交瘤。制备人杂交瘤的另一个问题是，出于伦理原因，难以获得用任意抗原免疫的B淋巴细胞。为了克服这个问题，已经开发了体外免疫方法，使大量B淋巴细胞能够接触培养的癌细胞或可溶性抗原。关于人杂交瘤的部分解释了人类融合亲本、体外免疫方法以及使用电融合方法制备人-人杂交瘤。最后，综述了人单克隆抗体在医学上的应用以及超天然单克隆抗体的制备。这些包括多功能单克隆抗体以及通过交换或修饰轻链而具有更高亲和力和特异性的改造单克隆抗体。
