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

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.

BRAIN Initiative: Cutting-Edge Tools and Resources for the Community

“…and colleagues (Gong et al, 2016) have pursued a multipronged approach to develop, validate, and enhance community access to a diverse toolbox of renewable recombinant antibodies and affinity reagents for neuroscience. First, they have converted a substantial portion of their existing library of mouse monoclonal antibodies (mAbs) into recombinant form (Andrews et al, 2019). This ensures permanent archiving in and streamlined distribution from plasmid banks and allows for more reliable expression than conventionally generated mAbs.…”

Implementation of an antibody characterization process: Application to the major ALS/FTD disease gene C9ORF72

“…The number of antibodies available for each target is variable with, for example, 8 antibodies for the newly identified ALS disease gene NEK1 and over 700 for SOD1, an abundant and well-studied ALS gene. If the number of antibodies is unmanageable, it would be advisable to limit validation studies to renewable antibodies such as monoclonals (Andrews et al, 2019). Also, whereas the cost of antibody purchase can appear to be a barrier, many companies have refund policies that can decrease costs if the antibodies to not meet reasonable functional criteria.…”

“…Antibodies are vital tools in the biomedical research arsenal and in keeping with their importance there are over a million unique antibodies that are commercially available (Dr. Matt Baker, ThermoFisher, personal communication). In addition are the outputs of numerous, publicly-funded projects, which have generated recombinant or monoclonal antibodies, thus creating renewable antibodies with the potential for high reproducibility (Hornsby et al, 2015;Marcon et al, 2015;Na et al, 2016;Venkataraman et al, 2018;Andrews et al, 2019). The largest public antibody initiative has generated greater than 24,000 antibodies corresponding to almost 17,000 human genes, with a focus on polyclonal antibodies (Uhlen et al, 2015).…”

A recombinant antibody toolbox for Dictyostelium discoideum

“…One easy solution is to sequence the antibody genes directly from the hybridoma cells. This guarantees a permanent, inexpensive and flexible storage, since all the information is contained in an electronic file, and/or in a DNA plasmid [10]. Once the sequence information is available, it can easily be stored and propagated, and it can also be used to produce the corresponding recombinant antibody.…”