Department of Biochemistry and Molecular Biology, Colorado State University, Fort Collins, United States.
Geneva Antibody Facility, Faculty of Medicine, University of Geneva, Geneva, Switzerland.
Elife. 2021 Dec 31;10:e72093. doi: 10.7554/eLife.72093.
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.
抗体是基础和转化生物科学研究中大量应用不可或缺的工具；然而，使用传统的动物产生的抗体存在一些缺点。这些缺点包括缺乏标准化导致重现性问题、从商业来源购买抗体的高成本，以及大量使用动物来产生抗体所涉及的伦理问题。为了解决这些问题，我们开发了用于产生低成本、高产量的重组单克隆抗体和针对主要序列蛋白表位的抗体片段的实用方法和工具。我们在这里描述了这些方法，以及多样化单克隆抗体的方法，包括定制抗体的物种特异性、产生遗传编码的小抗体片段，以及将单链抗体片段（例如 scFv）转化为全长、二价抗体。本研究重点介绍了针对有丝分裂和着丝粒功能重要表位的抗体；然而，这里描述的方法和试剂可适用于任何领域的抗体和抗体片段。
