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. 2011 Nov;59(5):975-83.
doi: 10.1111/j.1365-2559.2011.04034.x.

Human epidermal growth factor receptor 2 (HER2) immunoreactivity: specificity of three pharmacodiagnostic antibodies

Anne-Sofie Schrohl  1 , Hans Christian Pedersen, Sussie Steen Jensen, Signe Lykke Nielsen, Nils Brünner
Affiliations
Free PMC article

Human epidermal growth factor receptor 2 (HER2) immunoreactivity: specificity of three pharmacodiagnostic antibodies

Anne-Sofie Schrohl et al. Histopathology. 2011 Nov.
Free PMC article

Abstract

Aims: The availability of specific antibody-based test systems is essential to testing of HER2 protein expression. Here, we mapped epitopes recognized by three pharmacodiagnostic HER2 antibodies and investigated their specificity towards peptides and fusion proteins homologous to the intracellular domains of HER1, HER2, HER3 and HER4. The investigated antibodies were PATHWAY(®) HER2 (clone 4B5; Ventana Medical Systems Inc., Tucson, AZ, USA), HercepTest™ (Dako Denmark A/S, Glostrup, Denmark), and Oracle(®) HER2 (clone CB11; Leica Microsystems GmbH, Wetzlar, Germany).

Methods and results: Epitopes were mapped using the alanine scanning method. Specificity was investigated in immunohistochemical stainings, competitive enzyme-linked immunosorbent assay (ELISA) and immunoblotting. All three antibodies reacted with HER2 proteins and peptides in immunohistochemical stainings, ELISA and immunoblotting. PATHWAY(®) HER2 also stained HER4-expressing cells, reacted with HER4 peptide in ELISA and detected HER4 fusion protein in an immunoblot. Oracle(®) HER2 weakly detected HER4 in immunohistochemical stainings, whereas the HercepTest™ antibody showed no cross-reactivity with other HER proteins.

Conclusion: Our study shows that the PATHWAY(®) HER2 antibody can bind HER4 peptides and fusion proteins in three different experimental settings. This should be investigated further to determine whether binding of HER4 also occurs in tissue samples and if such binding would have implications for therapy decisions for breast cancer patients.

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Figures

Figure 1
Figure 1
Amino acids in human epidermal growth factor receptor 2 (HER2) that constitute epitopes recognized by each antibody (A) and alignment of part of the intracellular domains of HER1, HER2, HER3 and HER4 (B).
Figure 2
Figure 2
PATHWAY® human epidermal growth factor receptor 2 (HER2) immunostaining of Chinese hamster ovary (CHO) K1 cells transfected with the intracellular domain of HER1 (A), HER2 (B), HER3 (C) or HER4 (D). A representative area is shown.
Figure 3
Figure 3
HercepTest™ human epidermal growth factor receptor 2 (HER2) immunostaining of Chinese hamster ovary (CHO) K1 cells transfected with the intracellular domain of HER1 (A), HER2 (B), HER3 (C) or HER4 (D). A representative area is shown.
Figure 4
Figure 4
Oracle® human epidermal growth factor receptor 2 (HER2) immunostaining of Chinese hamster ovary (CHO) K1 cells transfected with the intracellular domain of HER1 (A), HER2 (B), HER3 (C) or HER4 (D). A representative area is shown.
Figure 5
Figure 5
Results of competitive enzyme-linked immunosorbent assay (ELISA). Human epidermal growth factor receptor 1 (HER1), HER2 and HER4 peptides were added and competition investigated for the PATHWAY® HER2 antibody (A), the HercepTest™ antibody (B) and the CB11 antibody concentrate (C). The figure shows the result of one experiment representative of three independent ones.
Figure 6
Figure 6
Immunoblots of glutathione S-transferase (GST)–human epidermal growth factor receptor (HER) fusion proteins. Identical blots were developed using the PATHWAY® HER2 antibody (A), the HercepTest™ antibody (B) and the CB11 antibody concentrate (C). D, The loading control (anti-GST). The figure shows the result of one experiment representative of three independent ones.
Figure 7
Figure 7
Visualization of glutathione S-transferase (GST)–human epidermal growth factor receptor 4 (HER4) after stripping and reprobing of the blot initially developed using the PATHWAY® HER2 antibody.
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