Abstract
Heparin-binding EGF-like growth factor (HB-EGF) is a member of the EGF family of growth factors that bind to and activate the EGF receptor (EGFR/ErbB1) and ErbB4. HB-EGF plays pivotal roles in pathophysiological processes, including cancer. Thus, monoclonal antibodies (mAbs) for HB-EGF detection could be an important tool in the therapeutic diagnosis of HB-EGF-related cancers and other diseases. However, few mAbs, especially those applicable for immunohistochemistry (IHC), have been established to date. In this study, we generated a clone of hybridoma-derived mAb 2-108 by immunizing mice with recombinant human HB-EGF protein expressed by human cells. The mAb 2-108 specifically bound to human HB-EGF but not to mouse HB-EGF and was successful in immunoblotting, even under reducing conditions, immunoprecipitation, and immunofluorescence for unfixed as well as paraformaldehyde-fixed cells. Notably, this mAb was effective in IHC of paraffin-embedded tumor specimens. Epitope mapping analysis showed that mAb 2-108 recognized the N-terminal prodomain in HB-EGF. These results indicate that this new anti-HB-EGF mAb 2-108 would be useful in the diagnosis of HB-EGF-related cancers and would be a strong tool in both basic and clinical research on HB-EGF.
Highlights
Heparin-binding EGF-like growth factor (HB-EGF) is a member of the EGF family of growth factors that bind to and activate the EGF receptor and ErbB4.(1,2) Like other members of the EGF family, HB-EGF is synthesized as a membrane-anchored protein, which is composed of a signal peptide, a propeptide, and heparinbinding, EGF-like, juxtamembrane, transmembrane, and cytoplasmic domains.[3] proHB-EGF is biologically active as a juxtacrine growth factor that signals to neighboring cells in a nondiffusible manner[4,5] and functions as the receptor for the diphtheria toxin (DT).(6,7) proHB-EGF is cleaved at its juxtamembrane domain by metalloproteases in a process called ectodomain shedding.[8]. Ectodomain shedding of proHB-EGF yields a soluble form of HB-EGF, which is a potent mitogen and chemoattractant for cells expressing the cognate ErbB receptor.[9,10]
We show that monoclonal antibodies (mAbs) 2-108 can be successfully used for IHC of paraffinembedded specimens as well as other applications
Previous studies have not isolated mAbs that recognize this region, here we obtained several hybridoma clones that produce mAbs that recognize this region of HB-EGF
Summary
Heparin-binding EGF-like growth factor (HB-EGF) is a member of the EGF family of growth factors that bind to and activate the EGF receptor and ErbB4.(1,2) Like other members of the EGF family, HB-EGF is synthesized as a membrane-anchored protein (proHB-EGF), which is composed of a signal peptide, a propeptide, and heparinbinding, EGF-like, juxtamembrane, transmembrane, and cytoplasmic domains.[3] proHB-EGF is biologically active as a juxtacrine growth factor that signals to neighboring cells in a nondiffusible manner[4,5] and functions as the receptor for the diphtheria toxin (DT).(6,7) proHB-EGF is cleaved at its juxtamembrane domain by metalloproteases in a process called ectodomain shedding.[8]. Monoclonal antibodies (mAbs) available for HB-EGF detection could be an important tool in the diagnosis of HB-EGF-related cancers and other diseases. A number of mAbs reacting to HB-EGF have been isolated,(23,27) those especially applicable in immunohistochemistry (IHC) of paraffin-embedded specimens have not been established. We generated mAbs to HB-EGF and obtained a clone of hybridoma that detects HB-EGF both in intact cells and fixed paraffin-embedded sections. We characterize this antibody and demonstrate its usefulness for several applications. Our results suggest that this new anti-HB-EGF mAb 2-108 would be a powerful tool in the therapeutic diagnosis of HB-EGF-related cancers and other diseases
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