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.
journal_name
Elifejournal_title
eLifeauthors
Andrews NP,Boeckman JX,Manning CF,Nguyen JT,Bechtold H,Dumitras C,Gong B,Nguyen K,van der List D,Murray KD,Engebrecht J,Trimmer JSdoi
10.7554/eLife.43322subject
Has Abstractpub_date
2019-01-22 00:00:00issn
2050-084Xpii
43322journal_volume
8pub_type
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