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.
All data generated during this study are included in the manuscript. We will also deposit the plasmid text files and maps on our institutional repository and AddGene.
- Jennifer G DeLuca
- Timothy J Stasevich
- Ning Zhao
- Dileep Varma
- Timothy J Stasevich
- Lori Sherman
- Steven M Anderson
The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.
- Silke Hauf, Virginia Tech, United States
© 2021, DeLuca et al.
This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.
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