Abstract
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.
Highlights
Antibodies are indispensable tools used in a diverse array of applications in the biomedical sciences including detection of biomolecules in cells, tissues, and biological fluids; protein purification; functional depletion of proteins from cells and cell extracts; medical diagnostics, and therapeutic medicine
While this study focuses on antibodies relevant to cell division and mitosis, these approaches are widely applicable for antibodies and antibody fragments across fields
We collected time-lapse images, and as shown in Figure 5F and Figure 5 – figure supplement 1, the genetically encoded pMELT scFv-GFP recognizes kinetochores in early mitosis when phosphorylation of the KNL1 MELT repeats is high, but much less so in late mitosis, when phosphorylation of the MELT repeats is low. These results demonstrate that small immunological probes can be generated from primary monoclonal antibody sequences and successfully expressed in mitotic cells to detect mitotic antigens, and importantly, post-translational protein modifications of mitotic targets in living cells
Summary
Antibodies are indispensable tools used in a diverse array of applications in the biomedical sciences including detection of biomolecules in cells, tissues, and biological fluids; protein purification; functional depletion of proteins from cells and cell extracts; medical diagnostics, and therapeutic medicine While these reagents are essential for almost all areas of research in the biosciences, there are drawbacks to using traditional antibodies generated in animals. With the primary amino acid sequence of an antibody in-hand, researchers can diversify the original reagent and create derivative tools such as antibody fragments, that can be genetically fused to other molecules, such as fluorophores, to generate custom tools with diverse functionalities. While this study focuses on antibodies relevant to cell division and mitosis, these approaches are widely applicable for antibodies and antibody fragments across fields
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