Steenbakkers P G, van Wezenbeek P M, Olijve W
Department of Immunology, Organon International B.V., Oss, Netherlands.
J Immunol Methods. 1993 Jul 6;163(1):33-40. doi: 10.1016/0022-1759(93)90236-z.
This paper reports the generation of monoclonal antibody producing hybridomas from a small number of antigen-specific B cells selected by panning on antigen-coated dishes and rosetting with antigen-coupled paramagnetic beads. Anti-HIV positive B cells from spleen could be recovered by panning with an efficiency of 5% and a purity of 24%. Immunobead selection of anti-HIV positive B cells from the same mice yielded a recovery of 17% and a purity of 7%. Various experimental conditions with respect to the selection of specific B cells were investigated, leading to an optimized protocol for the isolation of a limited subset of B cells. The selected cells retained their property to produce immunoglobulins and could be clonally expanded in the presence of human T cell supernatant and irradiated murine thymoma helper cells to generate sufficient cells for a mini-electrofusion with NS-1 myeloma cells. Up to 78 specific hybridomas could be generated from one anti-HIV positive B cell. An overall efficiency of specific B cell immortalization of up to 10% was obtained.
本文报道了通过在抗原包被的培养皿上淘选并与抗原偶联的顺磁珠进行花环试验，从少量抗原特异性B细胞中产生分泌单克隆抗体的杂交瘤。通过淘选可从脾脏中回收抗HIV阳性B细胞，回收率为5%，纯度为24%。从同一只小鼠中通过免疫珠选择抗HIV阳性B细胞，回收率为17%，纯度为7%。研究了关于特异性B细胞选择的各种实验条件，从而得出了一种优化的方案，用于分离有限的B细胞亚群。所选细胞保留了产生免疫球蛋白的特性，并且在人T细胞上清液和经辐照的鼠胸腺瘤辅助细胞存在的情况下能够进行克隆扩增，以产生足够的细胞用于与NS-1骨髓瘤细胞进行微量电融合。从一个抗HIV阳性B细胞中最多可产生78个特异性杂交瘤。特异性B细胞永生化的总体效率高达10%。
