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

Elife. 2019 Jan 22;8:e43322. doi: 10.7554/eLife.43322.

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.

Keywords: antibodies; brain; immunohistochemistry; mouse; neuroscience; rat.

Publication types

  • Research Support, N.I.H., Extramural

MeSH terms

  • Animals
  • Antibodies, Monoclonal / immunology*
  • Antibody Specificity
  • Brain / diagnostic imaging*
  • Brain / immunology
  • Enzyme-Linked Immunosorbent Assay
  • Humans
  • Hybridomas / immunology
  • Immunoglobulin G / immunology*
  • Immunohistochemistry*
  • Mice
  • Neurosciences / methods
  • Rats
  • Recombinant Proteins / immunology

Substances

  • Antibodies, Monoclonal
  • Immunoglobulin G
  • Recombinant Proteins