Generation and diversification of recombinant monoclonal antibodies
Keith F DeLuca,
Jeanne E Mick,
Amy H Ide,
Wanessa C Lima,
Lori Sherman,
Kristin L Schaller,
Steven M Anderson,
Ning Zhao,
Timothy J Stasevich,
Dileep Varma,
Jakob Nilsson,
Jennifer G DeLuca
Affiliations
Keith F DeLuca
Department of Biochemistry and Molecular Biology, Colorado State University, Fort Collins, United States
Jeanne E Mick
Department of Biochemistry and Molecular Biology, Colorado State University, Fort Collins, United States
Amy H Ide
Department of Biochemistry and Molecular Biology, Colorado State University, Fort Collins, United States
Wanessa C Lima
Geneva Antibody Facility, Faculty of Medicine, University of Geneva, Geneva, Switzerland
Lori Sherman
CU Cancer Center Cell Technologies Shared Resource, University of Colorado Cancer Center, Anschutz Medical Campus, Aurora, United States
Kristin L Schaller
Department of Pediatric Hematology, Oncology and Bone Marrow Transplant, University of Colorado Anschutz Medical Campus, Aurora, United States
Steven M Anderson
CU Cancer Center Cell Technologies Shared Resource, University of Colorado Cancer Center, Anschutz Medical Campus, Aurora, United States; Department of Pathology, University of Colorado Anschutz Medical Campus, Aurora, United States
Department of Biochemistry and Molecular Biology, Colorado State University, Fort Collins, United States
Timothy J Stasevich
Department of Biochemistry and Molecular Biology, Colorado State University, Fort Collins, United States; Cell Biology Center and World Research Hub Initiative, Tokyo Institute of Technology, Yokohama, Japan
Dileep Varma
Department of Cell and Developmental Biology, Feinberg School of Medicine, Northwestern University, Chicago, United States
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.
