A Toolbox of IgG Subclass-Switched Recombinant Monoclonal Antibodies for Enhanced Multiplex Immunolabeling of Brain

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 trea™ent. 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. This work was funded by NIH research grants U24 NS050606, R24 NS092991 and U24 NS109113 to J. S. Trimmer. We thank the current and former staff members of the UC Davis/NIH NeuroMab Facility and the Trimmer laboratory for their contributions and dedicated efforts, and Dr. Randall Stewart at the National Institute of Neurological Disorders and Stroke for support and helpful advice. We thank Dr. Gavin Wright of the Sanger Institute for his generous gift of the P1316 expression plasmid and helpful advice.