title: [Novel mutation-specific monoclonal E-cadherin antibodies make possible allele differentiation at the protein level in tumors].
abstract: Somatic deletion mutations in the cell adhesion molecule E-cadherin are present in almost 50% of diffuse type gastric cancer. We recently generated monoclonal antibodies against an in-frame deletion of exon 9. The aim of this study was to generate and characterize monoclonal antibodies against the second mutational hot spot, in-frame deletions of exon 8. Lou/C rats were immunized using a KLH-coupled peptide that represents a unique sequence generated by fusion of exon 7 and exon 9 from an E-cadherin deletion mutation lacking exon 8. Hybridoma supernatants were tested in a solid-phase immunoassay using BSA-coupled peptide. Positive reacting hybridomas were confirmed by Western Blots, FACS analysis, and immunohistochemistry of E-cadherin negative carcinoma cells that had been transfected with mutant and wild-type E-cadherin cDNA, respectively. In addition, routine formalin fixed and paraffin embedded tissues from gastric cancer patients were analyzed using both mutation-specific and commercial monoclonal antibodies against E-cadherin, including HECD-1 and AEC. Two hybridoma supernatants, termed E-cad delta 8-1, were selected that reacted with the mutant peptide used for immunization and gave strong signals in Western Blot and FACS analysis with cells expressing mutant E-cadherin lacking exon 8. Wild-type protein expressing cells only reacted with the commercial antibodies but not with the two selected hybridoma supernatants. In contrast to AEC, monoclonal antibody HECD-1 did not react with exon 8 deleted E-cadherin, suggesting that the previously unknown epitope for this often used monoclonal antibody is located at least in part within exon 8. Four gastric cancer specimens known to express mutated E-cadherin mRNA strongly reacted with both mutation-specific supernatants and with AEC monoclonal antibody but not with HECD-1. Taken together, we succeeded in generating monoclonal antibodies reacting with mutant E-cadherin protein lacking exon 8. Furthermore, using both HECD-1 and the new mutation-specific antibodies E-cadherin immunoreactivity can for the first time be evaluated in an allele-specific manner in archival tissues.
authors.affiliation: Klinikum rechts der Isar, Technische Universität, Institut für Pathologie, München.
authors.forename: I
authors.initials: I
authors.lastname: Becker
authors.forename: K F
authors.initials: KF
authors.lastname: Becker
authors.forename: E
authors.initials: E
authors.lastname: Kremmer
authors.forename: M
authors.initials: M
authors.lastname: Eulitz
authors.forename: G
authors.initials: G
authors.lastname: Handschuh
authors.forename: H
authors.initials: H
authors.lastname: Höfler
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal; D000918:Antibody Specificity; D014408:Biomarkers, Tumor / Q000032:analysis / Q000235:genetics; D015153:Blotting, Western; D015820:Cadherins / Q000032:analysis* / Q000235:genetics* / Q000276:immunology; D018076:DNA, Complementary; D005091:Exons; D005434:Flow Cytometry; D006801:Humans; D006825:Hybridomas; D051381:Rats; D017384:Sequence Deletion; D013274:Stomach Neoplasms / Q000235:genetics* / Q000473:pathology*; D014162:Transfection
chemical_list: D000911:Antibodies, Monoclonal; D014408:Biomarkers, Tumor; D015820:Cadherins; D018076:DNA, Complementary