Generation and diversification of recombinant monoclonal antibodies

• Published in: eLife Vol. 10
• Main Authors: DeLuca, Keith F, Mick, Jeanne E, Ide, Amy H, Lima, Wanessa C, Sherman, Lori, Schaller, Kristin L, Anderson, Steven M, Zhao, Ning, Stasevich, Timothy J, Varma, Dileep, Nilsson, Jakob, DeLuca, Jennifer G
• Format: Journal Article
• Language: English
• Published: England eLife Science Publications, Ltd 31.12.2021; eLife Sciences Publications Ltd; eLife Sciences Publications, Ltd
• Subjects: Amino acids; Antibodies, Monoclonal - immunology; antibody; Antigenic determinants; Biochemistry and Chemical Biology; Bispecific antibodies; Cell Biology; Cell division; Chromosomes; Cloning; Epitopes; Epitopes - immunology; Humans; Immunology; kinetochore; Light; Medical equipment and supplies industry; Medical research; Medical test kit industry; Mitosis; Monoclonal antibodies; nanobody; Peptides; Plasmids; Proteins; Reagents; Recombinant Proteins - immunology; Reproducibility; scFv; spindle; Standardization; Tools and Resources; United States; antibody; cell biology; chemical biology; scFv; biochemistry; kinetochore; nanobody; mitosis; human; spindle
• ISSN: 2050-084X; 2050-084X
• 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.