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Selective Production of monoclonal Antibodies Using a Specific Affinity between Avidin and Biotin

利用抗生物素蛋白和生物素之间的特异性亲和力选择性生产单克隆抗体

基本信息

批准号：
08672506
负责人：
TOMITA Masahiro
金额：
$ 1.47万
依托单位：
Mie University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
1996
资助国家：
日本
起止时间：
1996 至 1998
项目状态：
已结题

项目摘要

A new method for the production of monoclonal antibodies were developed. First, an antigen-avidin was prepared to select the B lymphocytes pre-immunized by the antigen. A bifunctional cross-linker, MBS (m-maleimidobenzoyl N-hydroxysuccinimide), was employed for this purpose. Lysine residues in the avidin and cysteine residues in the antigen were covalently linked by use of heterofunctional groups in MBS such as NHS (N-hydroxysuccinimide) and maleimide, respectively. On the one hand, myeloma cells were biotinylated by NHS-biotin. Finally, B lymphocyte-antigen-avidin and biotin-myeloma cell were combined to produce B lymphocyte-antigen-avidin-biotin-mycloma cell complex. This B lymphocyte-myeloma cell complex was then selectively fused by a pulsed electric field (PEF) with a few kVcm^<-1> for a duration in 10mus range. The fusion efficiency of this new method resulted in 20 to 30-fold higher than that obtained by poly(ethylene glycol) method, which is commonly used for the production of monoclonal antibodies.In the next step, a method for in vitro immunization was employed to further develop this new method. The advantage of the usage of in vitro system is that (I) it can markedly reduce the period for the immunization and the amount of an antigen, and (II) it enables to use the antigens which make adverse effects on in vivo system. After B lymphocytes immunized in vitro system were selected by antigen-biotin, they were combined with biotin-myeloma cells by use of a specific affinity between avidin and biotin. The B lymphocyte-antigen-biotin- streptavidin-biotin-mycloma cell complex was then fused by an electric field. In consequence, we could succeed in obtaining hybridoma cells which can produce monoclonal antibodies against the given antigen using not only in vivo system but also in vitro system with a newly developed PEF method.

建立了一种生产单克隆抗体的新方法。首先制备亲和抗原，选择经抗原预免疫的B淋巴细胞。双功能交联剂MBS（间马来酰亚胺苯甲酰基n -羟基琥珀酰亚胺）用于此目的。在MBS中，亲和素中的赖氨酸残基和抗原中的半胱氨酸残基分别通过异官能团如NHS （n -羟基琥珀酰亚胺）和马来酰亚胺共价连接。一方面，骨髓瘤细胞被nhs生物素生物素化。最后，B淋巴细胞-亲和抗原和生物素-骨髓瘤细胞结合形成B淋巴细胞-亲和抗原-生物素-骨髓瘤细胞复合物。B淋巴细胞-骨髓瘤细胞复合物在10mus范围内选择性地用几kVcm^<-1>的脉冲电场（PEF）融合持续时间。这种新方法的融合效率比通常用于生产单克隆抗体的聚乙二醇方法高20 ~ 30倍。下一步，我们将采用体外免疫的方法来进一步发展这种新方法。使用体外系统的优点是：(1)它可以显著缩短免疫周期和抗原的用量；(2)它可以利用对体内系统产生不利影响的抗原。抗原-生物素选择体外免疫系统的B淋巴细胞后，利用亲和素与生物素的特异性亲和力与生物素-骨髓瘤细胞结合。B淋巴细胞-抗原-生物素-链亲和素-生物素-骨髓瘤细胞复合物在电场作用下融合。因此，我们可以成功地在体内系统和体外系统中使用新开发的PEF方法获得能够产生针对给定抗原的单克隆抗体的杂交瘤细胞。