title: Activation state-specific monoclonal antibody detects tyrosine phosphorylated p185neu/erbB-2 in a subset of human breast tumors overexpressing this receptor.
abstract: Amplification of the neu/erbB-2/HER-2 gene and/or overexpression of its receptor tyrosine kinase product, p185neu/erbB-2 (p185), occurs in 15-40% of primary human breast tumors and is variably correlated with poor patient prognosis. Variability in predictive accuracy likely results from activation of p185 by agonist(s) in only a subset of tumors in which it is overexpressed, which may greatly affect outcomes. As a first step toward evaluating this hypothesis, we previously produced a polyclonal antibody that specifically recognizes the activated, tyrosine-phosphorylated forms of p185 and the closely related epidermal growth factor receptor (L. Bangalore et al., Proc. Natl. Acad. Sci. USA, 89: 11637-11641, 1992). We now describe the production of a mAb, PN2A, that specifically recognizes tyrosine-phosphorylated p185 and bears no cross-reactivity with closely related receptors. Furthermore, we demonstrate its reactivity in immunohistochemical staining of paraffin-embedded, formalin-fixed tumor samples. In a series of five p185-overexpressing human tumors examined thus far, PN2A reactivity was detected in two, indicating that p185 is phosphorylated and hence actively signaling in a subset. This reagent will facilitate both clinical and research analyses of p185 activity. Furthermore, this work serves as a prototype for similar analyses of other tyrosine phosphoproteins.
authors.affiliation: Department of Pathology, Yale University School of Medicine, New Haven, Connecticut 06510, USA.
authors.forename: M P
authors.initials: MP
authors.lastname: DiGiovanna
authors.forename: D F
authors.initials: DF
authors.lastname: Stern
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000096:biosynthesis / Q000276:immunology*; D000918:Antibody Specificity; D001943:Breast Neoplasms / Q000235:genetics / Q000378:metabolism / Q000648:ultrastructure*; D003429:Cross Reactions; D005260:Female; D015870:Gene Expression; D006801:Humans; D007150:Immunohistochemistry; D051379:Mice; D008969:Molecular Sequence Data; D010766:Phosphorylation; D018719:Erb-b2 Receptor Tyrosine Kinases / Q000032:analysis* / Q000276:immunology / Q000502:physiology; D015398:Signal Transduction / Q000502:physiology; D014407:Tumor Cells, Cultured; D014443:Tyrosine / Q000378:metabolism*
chemical_list: D000911:Antibodies, Monoclonal; D018719:Erb-b2 Receptor Tyrosine Kinases; D014443:Tyrosine