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

建立了一种制备单抗的新方法。首先，制备一种抗原-亲和素来筛选用该抗原预先免疫的B淋巴细胞。为此，采用了双官能团交联剂MBS(间马来酰亚胺基N-羟基琥珀酰亚胺)。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N-羟基丁二酰亚胺(NHS)和马来酰亚胺(Maleimide)等多官能团共价连接。一方面，骨髓瘤细胞被NHS-生物素生物素标记。最后，将B淋巴细胞-抗原-亲和素与生物素-骨髓瘤细胞结合，形成B淋巴细胞-抗原-亲和素-生物素-骨髓瘤细胞复合体。这个B淋巴细胞-骨髓瘤细胞复合体随后被脉冲电场(PEF)与几个kVcm~(-1)的脉冲电场选择性地融合在10mus范围内。这种新方法的融合效率比通常用于生产单抗的聚乙二醇法高20~30倍，下一步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时间和抗原的用量；(2)它可以使用对体内系统产生不利影响的抗原。体外免疫的B淋巴细胞经抗原-生物素筛选后，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。然后在电场作用下融合B淋巴细胞-抗原-生物素-链霉亲和素-生物素-粘液瘤细胞复合体。因此，我们不仅可以用体内系统，而且可以用新发展的PEF方法在体外系统中成功地获得能够产生抗给定抗原的单抗的杂交瘤细胞。

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.