Generation and diversification of recombinant monoclonal antibodies
- Colorado State University, United States
- University of Geneva, Switzerland
- University of Colorado Anschutz Medical Campus, United States
- Northwestern University, United States
- University of Copenhagen, Germany
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.
Data availability
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.
Article and author information
Author details
Funding
National Institute of General Medical Sciences (R35GM130365)
- Jennifer G DeLuca
National Institute of General Medical Sciences (MIRA R35GM119728)
- Timothy J Stasevich
National Institute of General Medical Sciences (K99GM141453)
- Ning Zhao
National Institute of General Medical Sciences (R01GM135391)
- Dileep Varma
National Science Foundation (MCB-1845761)
- Timothy J Stasevich
National Cancer Institute (P30CA046934)
- 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.
Reviewing Editor
- Silke Hauf, Virginia Tech, United States
Version history
- Received: July 10, 2021
- Preprint posted: September 11, 2021 (view preprint)
- Accepted: December 20, 2021
- Accepted Manuscript published: December 31, 2021 (version 1)
- Accepted Manuscript updated: January 7, 2022 (version 2)
- Version of Record published: January 17, 2022 (version 3)
Copyright
© 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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Generation and diversification of recombinant monoclonal antibodies
eLife 10:e72093.
https://doi.org/10.7554/eLife.72093
Further reading
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- Biochemistry and Chemical Biology
- Cell Biology
Antibodies are critical reagents to detect and characterize proteins. It is commonly understood that many commercial antibodies do not recognize their intended targets, but information on the scope of the problem remains largely anecdotal, and as such, feasibility of the goal of at least one potent and specific antibody targeting each protein in a proteome cannot be assessed. Focusing on antibodies for human proteins, we have scaled a standardized characterization approach using parental and knockout cell lines (Laflamme et al., 2019) to assess the performance of 614 commercial antibodies for 65 neuroscience-related proteins. Side-by-side comparisons of all antibodies against each target, obtained from multiple commercial partners, have demonstrated that: (i) more than 50% of all antibodies failed in one or more applications, (ii) yet, ~50–75% of the protein set was covered by at least one high-performing antibody, depending on application, suggesting that coverage of human proteins by commercial antibodies is significant; and (iii) recombinant antibodies performed better than monoclonal or polyclonal antibodies. The hundreds of underperforming antibodies identified in this study were found to have been used in a large number of published articles, which should raise alarm. Encouragingly, more than half of the underperforming commercial antibodies were reassessed by the manufacturers, and many had alterations to their recommended usage or were removed from the market. This first study helps demonstrate the scale of the antibody specificity problem but also suggests an efficient strategy toward achieving coverage of the human proteome; mine the existing commercial antibody repertoire, and use the data to focus new renewable antibody generation efforts.
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- Biochemistry and Chemical Biology
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An imbalance of the gut microbiota, termed dysbiosis, has a substantial impact on host physiology. However, the mechanism by which host deals with gut dysbiosis to maintain fitness remains largely unknown. In Caenorhabditis elegans, Escherichia coli, which is its bacterial diet, proliferates in its intestinal lumen during aging. Here, we demonstrate that progressive intestinal proliferation of E. coli activates the transcription factor DAF-16, which is required for maintenance of longevity and organismal fitness in worms with age. DAF-16 up-regulates two lysozymes lys-7 and lys-8, thus limiting the bacterial accumulation in the gut of worms during aging. During dysbiosis, the levels of indole produced by E. coli are increased in worms. Indole is involved in the activation of DAF-16 by TRPA-1 in neurons of worms. Our finding demonstrates that indole functions as a microbial signal of gut dysbiosis to promote fitness of the host.
