Detection of CD43 (Leukosialin) in colon adenoma and adenocarcinoma by novel monoclonal antibodies against its intracellular domain

Abstract

CD43 is a leukocyte-associated sialoglycoprotein which is also expressed in human colon adenoma and carcinoma. To obtain monoclonal antibodies (MAbs) that would react with CD43 in a glycosylation-independent way, antibodies were raised against a peptide corresponding to a portion of the CD43 cytoplasmic domain. Hybridomas were screened on paraffin sections from CD43- positive colon tumours. The reactivity of the antibodies with CD43 was verified by Western blot analysis of lysate of CHO cells transfected with human CD43 cDNA and by immunoprecipitation of lysates from CD43+ cell lines. Epitope mapping of antibodies was done using overlapping heptameric peptides. A detailed characterisation of one of the novel antibodies (CD43-3AI) is presented. This antibody reacts with the CD43 protein regardless of its glycosylation in Western blot analysis, immunoprecipitation and immunohistochemistry of paraffin sections. Immunohistochemical analysis of paraffin sections from colon adenoma and carcinoma tissues as well as colon cancer cell lines revealed that CD43 was predominantly localised intracellularly, in contrast to leukocyte-type cells. The MAb reacted more efficiently with paraffin-embedded colon adenoma and carcinoma cells than previously characterised CD43-specific antibodies. This should facilitate the evaluation of a potential role of CD43 during cancer development.