Generation and diversification of recombinant monoclonal antibodies

  1. Keith F DeLuca
  2. Jeanne E Mick
  3. Amy Hodges Ide
  4. Wanessa C Lima
  5. Lori Sherman
  6. Kristin L Schaller
  7. Steven M Anderson
  8. Ning Zhao
  9. Timothy J Stasevich
  10. Dileep Varma
  11. Jakob Nilsson
  12. Jennifer G DeLuca  Is a corresponding author
  1. Colorado State University, United States
  2. University of Geneva, Switzerland
  3. University of Colorado Anschutz Medical Campus, United States
  4. Northwestern University, United States
  5. 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

  1. Keith F DeLuca

    Department of Biochemistry and Molecular Biology, Colorado State University, Fort Collins, United States
    Competing interests
    No competing interests declared.
  2. Jeanne E Mick

    Department of Biochemistry and Molecular Biology, Colorado State University, Fort Collins, United States
    Competing interests
    No competing interests declared.
  3. Amy Hodges Ide

    Department of Biochemistry and Molecular Biology, Colorado State University, Fort Collins, United States
    Competing interests
    No competing interests declared.
  4. Wanessa C Lima

    Geneva Antibody Facility, Faculty of Medicine, University of Geneva, Geneva, Switzerland
    Competing interests
    No competing interests declared.
  5. Lori Sherman

    CU Cancer Center Cell Technologies Shared Resource, University of Colorado Anschutz Medical Campus, Aurora, United States
    Competing interests
    No competing interests declared.
  6. Kristin L Schaller

    Department of Pediatric Hematology, Oncology and Bone Marrow Transplant, University of Colorado Anschutz Medical Campus, Aurora, United States
    Competing interests
    No competing interests declared.
  7. Steven M Anderson

    Department of Pathology, University of Colorado Anschutz Medical Campus, Aurora, United States
    Competing interests
    No competing interests declared.
  8. Ning Zhao

    Department of Biochemistry and Molecular Biology, Colorado State University, Fort Collins, United States
    Competing interests
    No competing interests declared.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0001-7092-6229
  9. Timothy J Stasevich

    Department of Biochemistry and Molecular Biology, Colorado State University, Fort Collins, United States
    Competing interests
    No competing interests declared.
  10. Dileep Varma

    Department of Cell and Developmental Biology, Northwestern University, Chicago, United States
    Competing interests
    No competing interests declared.
  11. Jakob Nilsson

    The Novo Nordisk Foundation Center for Protein Research, University of Copenhagen, Copenhagen, Germany
    Competing interests
    No competing interests declared.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0003-4100-1125
  12. Jennifer G DeLuca

    Department of Biochemistry and Molecular Biology, Colorado State University, Fort Collins, United States
    For correspondence
    jdeluca@colostate.edu
    Competing interests
    Jennifer G DeLuca, Reviewing editor, eLife.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-3598-1721

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

  1. Silke Hauf, Virginia Tech, United States

Publication history

  1. Received: July 10, 2021
  2. Preprint posted: September 11, 2021 (view preprint)
  3. Accepted: December 20, 2021
  4. Accepted Manuscript published: December 31, 2021 (version 1)
  5. Accepted Manuscript updated: January 7, 2022 (version 2)
  6. 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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  1. Keith F DeLuca
  2. Jeanne E Mick
  3. Amy Hodges Ide
  4. Wanessa C Lima
  5. Lori Sherman
  6. Kristin L Schaller
  7. Steven M Anderson
  8. Ning Zhao
  9. Timothy J Stasevich
  10. Dileep Varma
  11. Jakob Nilsson
  12. Jennifer G DeLuca
(2021)
Generation and diversification of recombinant monoclonal antibodies
eLife 10:e72093.
https://doi.org/10.7554/eLife.72093
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