A toolbox of IgG subclass-switched recombinant monoclonal antibodies for enhanced multiplex immunolabeling of brain

Abstract

Generating recombinant monoclonal antibodies (R-mAbs) from mAb-producing hybridomas offers numerous advantages that increase the effectiveness, reproducibility, and transparent reporting of research. We report here the generation of a novel resource in the form of a library of recombinant R-mAbs validated for neuroscience research. We cloned immunoglobulin G (IgG) variable domains from cryopreserved hybridoma cells and input them into an integrated pipeline for expression and validation of functional R-mAbs. To improve efficiency over standard protocols, we eliminated aberrant Sp2/0-Ag14 hybridoma-derived variable light transcripts using restriction enzyme treatment. Further, we engineered a plasmid backbone that allows for switching of the IgG subclasses without altering target binding specificity to generate R-mAbs useful in simultaneous multiplex labeling experiments not previously possible. The method was also employed to rescue IgG variable sequences and generate functional R-mAbs from a non-viable cryopreserved hybridoma. All R-mAb sequences and plasmids will be archived and disseminated from open source suppliers.

Data availability

Plasmids and R-mAb sequences will be made available via Addgene (https://www.addgene.org/James_Trimmer/).

Article and author information

Author details

  1. Nicolas P Andrews

    Department of Neurobiology, Physiology and Behavior, University of California, Davis, Davis, United States
    Competing interests
    The authors declare that no competing interests exist.
  2. Justin X Boeckman

    Department of Neurobiology, Physiology and Behavior, University of California, Davis, Davis, United States
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-0022-1474
  3. Colleen F Manning

    Department of Neurobiology, Physiology and Behavior, University of California, Davis, Davis, United States
    Competing interests
    The authors declare that no competing interests exist.
  4. Joe T Nguyen

    Department of Molecular and Cellular Biology, University of California, Davis, Davis, United States
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-6647-0561
  5. Hannah Bechtold

    Department of Molecular and Cellular Biology, University of California, Davis, Davis, United States
    Competing interests
    The authors declare that no competing interests exist.
  6. Camelia Dumitras

    Department of Neurobiology, Physiology and Behavior, University of California, Davis, Davis, United States
    Competing interests
    The authors declare that no competing interests exist.
  7. Belvin Gong

    Department of Neurobiology, Physiology and Behavior, University of California, Davis, Davis, United States
    Competing interests
    The authors declare that no competing interests exist.
  8. Kimberly Nguyen

    Department of Neurobiology, Physiology and Behavior, University of California, Davis, Davis, United States
    Competing interests
    The authors declare that no competing interests exist.
  9. Deborah van der List

    Department of Neurobiology, Physiology and Behavior, University of California, Davis, Davis, United States
    Competing interests
    The authors declare that no competing interests exist.
  10. Karl D Murray

    Department of Neurobiology, Physiology and Behavior, University of California, Davis, Davis, United States
    Competing interests
    The authors declare that no competing interests exist.
  11. JoAnne Engebrecht

    Department of Physiology and Membrane Biology, University of California, Davis, Davis, United States
    Competing interests
    The authors declare that no competing interests exist.
  12. James S Trimmer

    Department of Neurobiology, Physiology and Behavior, University of California, Davis, Davis, United States
    For correspondence
    jtrimmer@ucdavis.edu
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-6117-3912

Funding

National Institute of Neurological Disorders and Stroke (U24 NS050606)

  • James S Trimmer

National Institute of Neurological Disorders and Stroke (R24 NS092991)

  • James S Trimmer

National Institute of Neurological Disorders and Stroke (U24 NS109113)

  • James S Trimmer

The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

Ethics

Animal experimentation: This study was performed in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. All of the animals were handled according to approved institutional animal care and use committee (IACUC) protocols (#20485) of the University of California Davis. The protocol was approved by the Committee on the Ethics of Animal Experiments of the University of California Davis (Animal Welfare Assurance Number A-3433-01). All procedures were performed under sodium pentobarbital anesthesia, and every effort was made to minimize suffering.

Reviewing Editor

  1. Richard Aldrich, The University of Texas at Austin, United States

Publication history

  1. Received: November 2, 2018
  2. Accepted: January 21, 2019
  3. Accepted Manuscript published: January 22, 2019 (version 1)
  4. Version of Record published: February 15, 2019 (version 2)

Copyright

© 2019, Andrews 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.

Metrics

  • 5,553
    Page views
  • 730
    Downloads
  • 11
    Citations

Article citation count generated by polling the highest count across the following sources: Crossref, PubMed Central, Scopus.

Download links

A two-part list of links to download the article, or parts of the article, in various formats.

Downloads (link to download the article as PDF)

Open citations (links to open the citations from this article in various online reference manager services)

Cite this article (links to download the citations from this article in formats compatible with various reference manager tools)

  1. Nicolas P Andrews
  2. Justin X Boeckman
  3. Colleen F Manning
  4. Joe T Nguyen
  5. Hannah Bechtold
  6. Camelia Dumitras
  7. Belvin Gong
  8. Kimberly Nguyen
  9. Deborah van der List
  10. Karl D Murray
  11. JoAnne Engebrecht
  12. James S Trimmer
(2019)
A toolbox of IgG subclass-switched recombinant monoclonal antibodies for enhanced multiplex immunolabeling of brain
eLife 8:e43322.
https://doi.org/10.7554/eLife.43322
  1. Further reading

Further reading

    1. Neuroscience
    Mingchao Yan et al.
    Tools and Resources

    Resolving trajectories of axonal pathways in the primate prefrontal cortex remains crucial to gain insights into higher-order processes of cognition and emotion, which requires a comprehensive map of axonal projections linking demarcated subdivisions of prefrontal cortex and the rest of brain. Here, we report a mesoscale excitatory projectome issued from the ventrolateral prefrontal cortex (vlPFC) to the entire macaque brain by using viral-based genetic axonal tracing in tandem with high-throughput serial two-photon tomography, which demonstrated prominent monosynaptic projections to other prefrontal areas, temporal, limbic, and subcortical areas, relatively weak projections to parietal and insular regions but no projections directly to the occipital lobe. In a common 3D space, we quantitatively validated an atlas of diffusion tractography-derived vlPFC connections with correlative green fluorescent protein-labeled axonal tracing, and observed generally good agreement except a major difference in the posterior projections of inferior fronto-occipital fasciculus. These findings raise an intriguing question as to how neural information passes along long-range association fiber bundles in macaque brains, and call for the caution of using diffusion tractography to map the wiring diagram of brain circuits.

    1. Medicine
    2. Neuroscience
    Simon Oxenford et al.
    Tools and Resources

    Background: Deep Brain Stimulation (DBS) electrode implant trajectories are stereotactically defined using preoperative neuroimaging. To validate the correct trajectory, microelectrode recordings (MER) or local field potential recordings (LFP) can be used to extend neuroanatomical information (defined by magnetic resonance imaging) with neurophysiological activity patterns recorded from micro- and macroelectrodes probing the surgical target site. Currently, these two sources of information (imaging vs. electrophysiology) are analyzed separately, while means to fuse both data streams have not been introduced.

    Methods: Here we present a tool that integrates resources from stereotactic planning, neuroimaging, MER and high-resolution atlas data to create a real-time visualization of the implant trajectory. We validate the tool based on a retrospective cohort of DBS patients (𝑁 = 52) offline and present single use cases of the real-time platform. Results: We establish an open-source software tool for multimodal data visualization and analysis during DBS surgery. We show a general correspondence between features derived from neuroimaging and electrophysiological recordings and present examples that demonstrate the functionality of the tool.

    Conclusions: This novel software platform for multimodal data visualization and analysis bears translational potential to improve accuracy of DBS surgery. The toolbox is made openly available and is extendable to integrate with additional software packages.

    Funding: Deutsche Forschungsgesellschaft (410169619, 424778381), Deutsches Zentrum für Luftund Raumfahrt (DynaSti), National Institutes of Health (2R01 MH113929), Foundation for OCD Research (FFOR).