Target-selective joint polymerase chain reaction: a robust and rapid method for high-throughput production of recombinant monoclonal antibodies from single cells.
Target-selective joint polymerase chain reaction: a robust and rapid method for high-throughput production of recombinant monoclonal antibodies from single cells.
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目标选择性关节聚合酶链反应：一种可靠的快速方法，用于高通量产生单个细胞的重组单克隆抗体。
DOI：
10.1186/1472-6750-11-75
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发表时间：
2011-07-21
影响因子：
3.5
通讯作者：
Isobe M
中科院分区：
文献类型：
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作者：
Yoshioka M;Kurosawa N;Isobe M
在治疗性抗体的开发过程中，需要大量的单克隆抗体来筛选最适合所需活性的单克隆抗体。虽然单细胞免疫球蛋白可变区基因克隆技术是一个强大的工具，但目前的方法仍然是快速生产大量重组抗体的障碍。我们已经开发了一种新的重叠延伸聚合酶链反应，目标选择性联合聚合酶链反应（TS-jPCR），并将其应用于线性免疫球蛋白基因表达构建体的生成。使用PCR扩增的免疫球蛋白可变基因和免疫球蛋白基因选择性盒（Ig盒）进行TS-jPCR，该免疫球蛋白基因选择性盒含有抗体表达的所有必需元件和免疫球蛋白基因特异性同源性的重叠区域。TS-jPCR技术简单且特异;即使在存在非特异性扩增的DNA的情况下，3 '-随机核苷酸加尾的免疫球蛋白可变基因片段和Ig盒也被组装成线性免疫球蛋白表达构建体。我们还开发了一种机器人磁珠处理仪器，用于基于单细胞的cDNA合成，以通过快速扩增5' cDNA末端PCR扩增免疫球蛋白可变基因。使用这些方法，我们能够在四天内从大量的单个浆细胞中产生重组单克隆抗体。我们的系统通过在短时间内快速生产大量重组单克隆抗体，减轻了抗体发现和工程改造的负担。
During the development of a therapeutic antibody, large numbers of monoclonal antibodies are required to screen for those that are best suited for the desired activity. Although the single cell-based immunoglobulin variable gene cloning technique is a powerful tool, the current methods remain an obstacle to the rapid production of large numbers of recombinant antibodies. We have developed a novel overlap extension polymerase chain reaction, the target-selective joint polymerase chain reaction (TS-jPCR), and applied it to the generation of linear immunoglobulin gene expression constructs. TS-jPCR is conducted using a PCR-amplified immunoglobulin variable gene and an immunoglobulin gene-selective cassette (Ig-cassette) that contains all essential elements for antibody expression and overlapping areas of immunoglobulin gene-specific homology. The TS-jPCR technique is simple and specific; the 3'-random nucleotide-tailed immunoglobulin variable gene fragment and the Ig-cassette are assembled into a linear immunoglobulin expression construct, even in the presence of nonspecifically amplified DNA. We also developed a robotic magnetic beads handling instrument for single cell-based cDNA synthesis to amplify immunoglobulin variable genes by rapid amplification of 5' cDNA ends PCR. Using these methods, we were able to produce recombinant monoclonal antibodies from large numbers of single plasma cells within four days. Our system reduces the burden of antibody discovery and engineering by rapidly producing large numbers of recombinant monoclonal antibodies in a short period of time.
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影响因子：
14.9
作者：
Hamilton MD;Nuara AA;Gammon DB;Buller RM;Evans DH
通讯作者：
Evans DH
影响因子：
64.8
作者：
Wrammert, Jens;Smith, Kenneth;Wilson, Patrick C.
通讯作者：
Wilson, Patrick C.
影响因子：
2.2
作者：
Tiller, Thomas;Busse, Christian E.;Wardemann, Hedda
通讯作者：
Wardemann, Hedda
影响因子：
9.2
作者：
Liu, QH;Li, MZ;Elledge, SJ
通讯作者：
Elledge, SJ
DOI：
10.1038/nri2744
发表时间：
2010-05
期刊：
Nature reviews. Immunology
影响因子：
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作者：
通讯作者：
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