Generation and diversification of recombinant monoclonal antibodies.
Generation and diversification of recombinant monoclonal antibodies.
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DOI：
10.7554/elife.72093
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发表时间：
2021-12-31
期刊：
影响因子：
7.7
通讯作者：
DeLuca JG
中科院分区：
文献类型：
--
作者：
DeLuca KF;Mick JE;Ide AH;Lima WC;Sherman L;Schaller KL;Anderson SM;Zhao N;Stasevich TJ;Varma D;Nilsson J;DeLuca JG
抗体是在基础和翻译生物科学研究中大量使用的不可或缺的工具；然而，使用传统的动物产生的抗体存在缺陷。这些问题包括缺乏标准化导致重复性问题、从商业来源购买抗体的高昂成本，以及对用于产生抗体的大量动物的伦理担忧。为了解决这些问题，我们开发了实用的方法和工具，用于从初级序列中产生针对蛋白质表位的重组单抗和抗体片段的低成本、高产量的制剂。我们在这里描述了这些方法，以及使单抗多样化的方法，包括定制抗体种类特异性，产生遗传编码的小抗体片段，以及将单链抗体片段(例如单链抗体片段)转化为全长双价抗体。这项研究的重点是针对对有丝分裂和动粒功能重要的表位的抗体；然而，这里描述的方法和试剂适用于任何领域使用的抗体和抗体片段。
Antibodies are indispensable tools used for a large number of applications in both foundational and translational bioscience research; however, there are drawbacks to using traditional antibodies generated in animals. These include a lack of standardization leading to problems with reproducibility, high costs of antibodies purchased from commercial sources, and ethical concerns regarding the large number of animals used to generate antibodies. To address these issues, we have developed practical methodologies and tools for generating low-cost, high-yield preparations of recombinant monoclonal antibodies and antibody fragments directed to protein epitopes from primary sequences. We describe these methods here, as well as approaches to diversify monoclonal antibodies, including customization of antibody species specificity, generation of genetically encoded small antibody fragments, and conversion of single chain antibody fragments (e.g. scFv) into full-length, bivalent antibodies. This study focuses on antibodies directed to epitopes important for mitosis and kinetochore function; however, the methods and reagents described here are applicable to antibodies and antibody fragments for use in any field.