Cloning single-chain antibody fragments (ScFv) from hyrbidoma cells.
Cloning single-chain antibody fragments (ScFv) from hyrbidoma cells.
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DOI：
10.1007/978-1-61779-974-7_3
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发表时间：
2012
影响因子：
--
通讯作者：
L. Toleikis;A. Frenzel
中科院分区：
文献类型：
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作者：
L. Toleikis;A. Frenzel
尽管通过噬菌体展示等技术产生抗体的影响越来越大，但杂交瘤技术仍然为产生针对不同靶点的高亲和力结合物提供了有价值的工具。但是使用杂交瘤来源的抗体存在一些局限性。杂交瘤克隆的来源多为大鼠或小鼠b淋巴细胞。因此，人抗小鼠或人抗大鼠抗体反应可能导致这些抗体的免疫原性。这导致这些抗体的人源化的必要性，其中蛋白质的氨基酸序列的知识是不可剥夺的。此外，其他体外修饰，如亲和成熟或与其他蛋白质的融合，依赖于抗原结合结构域的克隆。本文介绍了从杂交瘤细胞中分离RNA、扩增VL和VH的引物以及scFv格式抗体的克隆及其在大肠杆菌中的表达。
Despite the rising impact of the generation of antibodies by phage display and other technologies, hybridoma technology still provides a valuable tool for the generation of high-affinity binders against different targets. But there exist several limitations of using hybridoma-derived antibodies. The source of the hybridoma clones are mostly rat or mouse B-lymphocytes. Therefore a human-anti-mouse or human-anti-rat antibody response may result in immunogenicity of these antibodies. This leads to the necessity of humanization of these antibodies where the knowledge of the amino acid sequence of the proteins is inalienable. Furthermore, additional in vitro modifications, e.g., affinity maturation or fusion to other proteins, are dependent on cloning of the antigen-binding domains.Here we describe the isolation of RNA from hybridoma cells and the primers that can be used for the amplification of VL and VH as well as the cloning of the antibody in scFv format and its expression inEscherichia coli.