A toolbox of IgG subclass-switched recombinant monoclonal antibodies for enhanced multiplex immunolabeling of brain
- University of California, Davis, United States
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
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.
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.
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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
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