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

Author:

Andrews Nicolas P1,Boeckman Justin X1ORCID,Manning Colleen F1,Nguyen Joe T2ORCID,Bechtold Hannah2,Dumitras Camelia1,Gong Belvin1,Nguyen Kimberly1,van der List Deborah1,Murray Karl D1,Engebrecht JoAnne2,Trimmer James S13ORCID

Affiliation:

1. Department of Neurobiology, Physiology and Behavior, University of California, Davis, United States

2. Department of Molecular and Cellular Biology, University of California, Davis, United States

3. Department of Physiology and Membrane Biology, 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.

Funder

National Institute of Neurological Disorders and Stroke

Publisher

eLife Sciences Publications, Ltd

Subject

General Immunology and Microbiology,General Biochemistry, Genetics and Molecular Biology,General Medicine,General Neuroscience

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