Benefits and pitfalls of secondary antibodies: Why choosing the right secondary is of primary importance

Colleen F. Manning, Angeliki M. Bundros, James Trimmer

Research output: Contribution to journalArticle

28 Scopus citations

Abstract

Simultaneous labeling of multiple targets in a single sample, or multiplexing, is a powerful approach to directly compare the amount, localization and/or molecular properties of different targets in the same sample. Here we highlight the robust reliability of the simultaneous use of multiple mouse monoclonal antibodies (mAbs) of different immunoglobulin G (IgG) subclasses in a wide variety of multiplexing applications employing anti-mouse IgG subclass-specific secondary antibodies (2°Abs). We also describe the unexpected finding that IgG subclass-specific 2°Abs are superior to general anti-mouse IgG 2°Abs in every tested application in which mouse mAbs were used. This was due to a detection bias of general anti-mouse IgG-specific 2°Abs against mAbs of the most common mouse IgG subclass, IgG1, and to a lesser extent IgG2b mAbs. Thus, when using any of numerous mouse mAbs available through commercial and non-profit sources, for cleaner and more robust results each mAb should be detected with its respective IgG subclass-specific 2°Ab and not a general anti-mouse IgG-specific 2°Ab.

Original languageEnglish (US)
Article numbere38313
JournalPLoS One
Volume7
Issue number6
DOIs
StatePublished - Jun 1 2012

ASJC Scopus subject areas

  • Agricultural and Biological Sciences(all)
  • Biochemistry, Genetics and Molecular Biology(all)
  • Medicine(all)

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