Hapten-Anti-Hapten Technique for Two-Color IHC Detection of Phosphorylated EGFR and H2AX Using Primary Antibodies Raised in the Same Host Species

Methods Mol Biol. 2017;1554:155-160. doi: 10.1007/978-1-4939-6759-9_8.


Multiplex staining of cell and tissue sections with antibodies raised in the same host species is a serious challenge because of unwanted but inevitable cross-reactivity of secondary antibodies with irrelevant primary antibodies. Several techniques can be used to overcome this obstacle including direct labeling of primary antibodies with fluorescent tags and using tyramide signal amplification. Unfortunately these techniques either lack sensitivity, or require a long multistep protocol which can cause physical damage of specimens. As an alternative, we have developed a protocol based on conjugation of primary antibodies to small-size hapten molecules which can be detected with hapten-specific fluorescent secondary antibodies. This technique has been used for two-color labeling of Y845 phosphorylated Epidermal Growth Factor Receptor (EGFR) and S139 phosphorylated histone H2AX protein in A431 human epidermoid carcinoma cells. Our novel hapten-anti-hapten detection chemistry allows for generating a stronger fluorescent signal and completely avoid cross-interactions of secondary antibodies with irrelevant primary antibodies.

Keywords: Anti-hapten secondary antibodies; Hapten-conjugated primary antibodies; Phospho-specific antibodies; Phosphorylated EGFR; Phosphorylated H2AX; Two-color immunofluorescence.

MeSH terms

  • Antibodies / immunology*
  • Antibody Specificity / immunology
  • Cell Line
  • ErbB Receptors / metabolism*
  • Fluorescent Antibody Technique*
  • Haptens / immunology*
  • Histones / metabolism*
  • Humans
  • Immunoconjugates*
  • Immunohistochemistry / methods*
  • Microscopy, Fluorescence
  • Phosphorylation
  • Species Specificity


  • Antibodies
  • Haptens
  • Histones
  • Immunoconjugates
  • ErbB Receptors