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

Nicolas P. Andrews, Justin X. Boeckman, Colleen F. Manning, Joe T. Nguyen, Hannah Bechtold, Camelia Dumitras, Belvin Gong, Kimberly Nguyen, Deborah van der List, Karl D Murray, Jo Anne Engebrecht, James Trimmer

Research output: Contribution to journalArticle

5 Scopus citations

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.

Original languageEnglish (US)
JournaleLife
Volume8
DOIs
StatePublished - Jan 22 2019

Keywords

  • antibodies
  • brain
  • immunohistochemistry
  • mouse
  • neuroscience
  • rat

ASJC Scopus subject areas

  • Neuroscience(all)
  • Biochemistry, Genetics and Molecular Biology(all)
  • Immunology and Microbiology(all)

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    Andrews, N. P., Boeckman, J. X., Manning, C. F., Nguyen, J. T., Bechtold, H., Dumitras, C., Gong, B., Nguyen, K., van der List, D., Murray, K. D., Engebrecht, J. A., & Trimmer, J. (2019). A toolbox of IgG subclass-switched recombinant monoclonal antibodies for enhanced multiplex immunolabeling of brain. eLife, 8. https://doi.org/10.7554/eLife.43322