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

ELife
Nicolas P AndrewsJames S Trimmer

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.

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Citations

Aug 15, 2019·Nucleic Acids Research·Wanessa C LimaPierre Cosson
Apr 12, 2020·BMC Research Notes·Wanessa C LimaPierre Cosson
Nov 6, 2020·Current Protocols in Neuroscience·James S Trimmer
Nov 14, 2020·Current Protocols in Neuroscience·Clara E Gavira-O'NeillJames S Trimmer
May 27, 2021·Nature·Delphine AuthemanGavin J Wright

Methods Mentioned

BETA
Protein Capture
PCR
restriction digest
electrophoresis
transfection

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