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bioRxiv · 10.1101/483537

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

Andrews, N. P.·Boeckman, J. X.·Manning, C.·Nguyen, J. T.·Bechtold, H.·Dumitras, C.·Gong, B.·Nguyen, K.·van der List, D.·Murray, K. D.·Engebrecht, J.·Trimmer, J.
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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 treaent. 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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Andrews, N. P., Boeckman, J. X., Manning, C., Nguyen, J. T., Bechtold, H., Dumitras, C., Gong, B., Nguyen, K., van der List, D., Murray, K. D., Engebrecht, J., Trimmer, J.. 2018-11-30. A toolbox of IgG subclass-switched recombinant monoclonal antibodies for enhanced multiplex immunolabeling of brain. https://doi.org/10.1101/483537

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