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

PLoS One. 2012;7(6):e38313. doi: 10.1371/journal.pone.0038313. Epub 2012 Jun 1.

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.

Publication types

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

MeSH terms

  • Animals
  • Antibodies, Monoclonal / immunology*
  • Antibody Specificity / immunology
  • Brain / cytology
  • Brain / metabolism
  • Fluorescent Antibody Technique
  • Humans
  • Immunoassay / methods*
  • Immunoblotting
  • Immunoglobulin Heavy Chains / immunology
  • Immunoglobulin Isotypes / immunology
  • Immunoglobulin Light Chains / immunology
  • Mice
  • Mice, Knockout
  • Rats

Substances

  • Antibodies, Monoclonal
  • Immunoglobulin Heavy Chains
  • Immunoglobulin Isotypes
  • Immunoglobulin Light Chains