A novel strategy for generation of monoclonal antibodies from single B cells using RT-PCR technique and in vitro expression
A novel strategy for generation of monoclonal antibodies from single B cells using RT-PCR technique and in vitro expression
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DOI：
10.1021/bp060092h
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发表时间：
2006-08-01
影响因子：
2.9
通讯作者：
Nakano, Hideo
中科院分区：
文献类型：
--
作者：
Jiang, XiuPing;Suzuki, Hirotatsu;Nakano, Hideo
单克隆抗体（Mab）是免疫学中重要的生物分子，在预后、诊断和治疗中具有广泛的应用。在这里，我们描述了一种新方法，称为(s) under bar ingle-(c) under bar ell (R) under barT-PCR-linked in IV (ex) under barpression (SICREX)，该方法能够高通量生成和筛选单克隆抗体。该方法需要使用与 B 细胞选择性标记物抗 CD19 结合的磁性微珠从免疫小鼠脾脏或人外周血中分离 B 细胞。通过RT-PCR分别扩增每个细胞的轻链(Lc)和重链(Hc)基因的Fd部分，然后通过重叠PCR技术与T7启动子、核糖体结合位点(rbs)和T7终止子的序列组合。来自单个 B 细胞的 Lc 和 Hc 基因的配对全长 DNA 片段由大肠杆菌体外转录和翻译系统同时表达，然后通过酶联免疫吸附测定来寻找对抗原具有亲和力的阳性片段。从用钙结合蛋白40免疫的小鼠的脾细胞中，获得针对抗原的K-d为1.6(+/-0.3)×10(-8)的Fab片段。以类似的方式从人外周血中获得针对 B-BSA 血型的 Fab 片段。 SICREX 方法简单、快速且通用，可以高通量生成来自各种动物来源的具有抗原结合活性的天然配对的 Lc 和 Hc。
Monoclonal antibodies (Mabs) are important biomolecules in immunology and have widespread applications in prognosis, diagnosis, and therapeutics. Here, we describe a novel approach called (s) under bar ingle-(c) under bar ell (R) under barT-PCR-linked in vitro (ex) under bar pression (SICREX), which enables the high-throughput generation and screening of Mabs. This approach entails the isolation of B cells from immunized mouse spleen or human peripheral blood using magnetic microbeads conjugated with a B-cell-selective marker, anti-CD19. The light chain (Lc) and Fd portion of heavy-chain (Hc) genes of each cell are separately amplified by RT-PCR and then combined with the sequences of a T7 promoter, a ribosome binding site (rbs), and a T7 terminator by an overlapping PCR technique. The paired full-length DNA fragments of Lc and Hc genes from single B cells are simultaneously expressed by an Escherichia coli in vitro transcription and translation system followed by an enzyme-linked immunosorbent assay to find positive fragments possessing the affinity for the antigen. From spleen cells of an immunized mouse with calcium binding protein 40, a Fab fragment with K-d of 1.6 (+/- 0.3) x 10(-8) against the antigen was obtained. From human peripheral blood, Fab fragments against a blood group B-BSA were obtained in a similar manner. The SICREX approach is simple, rapid and versatile, allowing the high-throughput generation of naturally paired Lc and Hc with antigen-binding activity from various animal sources.