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.
Highlights
IntroductionEnhancing the research community’s access to extensively validated Abs remains an important goal of antibody developers in both academia and industry (Taussig et al, 2018)
Antibodies (Abs) are the workhorses of biomedical research
We developed a coherent pipeline of protocols for effective cloning of intact recombinant monoclonal antibodies (mAbs) (R-mAbs) from cryopreserved hybridoma cells and their subsequent validation compared to their parent mAbs
Summary
Enhancing the research community’s access to extensively validated Abs remains an important goal of antibody developers in both academia and industry (Taussig et al, 2018). This has been the topic of a number of recent conferences and commentaries, led to changes in journal practices as related to transparent reporting of antibody-based research including the use of Research Resource Identifiers or RRIDs, and resulted in large-scale NIH and EU Ab development initiatives (e.g., the NIH Common Fund Protein Capture Reagent Program, the EU Affinomics Program).
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