Monoclonal antibody specific for α2→8-linked oligo deaminated neuraminic acid (KDN) sequences in glycoproteins. Preparation and characterization of a monoclonal antibody and its application in immunohistochemistry |
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| Authors: | A Kanamori K Kitajima Y Inoue S Inoue Z Xulei C Zuber J Roth J Ye F A Troy II |
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| Institution: | (1) Department of Biophysics and Biochemistry, Graduate School of Science, University of Tokyo, Hongo-7, 113 Tokyo, Japan;(2) School of Pharmaceutical Sciences, Showa University, Hatanodai-1, 142 Tokyo, Japan;(3) Division of Cell and Molecular Pathology, Department of Pathology, University of Zürich, Schmelzbergstrasse 12, CH-8091 Zürich, Switzerland;(4) Department of Biological Chemistry, School of Medicine, University of California, 95616 Davis, CA, USA |
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| Abstract: | Two particular types of sialoglycoproteins have been detected in fish: polysialoglycoproteins containing  2 8-linked polysialic acid (8Neu5Gc2)
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present in unfertilized Salmonidae fish eggs, and glycoproteins bearing oligo/polymers of deaminated neuraminic acids (KDN) found in the vitelline envelope of the eggs and ovarian fluid. We report the preparation and characterization of a monoclonal antibody specifically recognizing oligo/polymers of KDN sequences in glycoproteins and its application in immunohistochemistry. Fusion of spleen cells from a BALB/c mouse immunized with a KDN-rich glycoprotein (KDN-gp) containing (8KDN2)
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6(KDN23Gal 13GlNAc13) GalNAc1 residues, with mouse myeloma cells yielded a hybrid cell line producing a monoclonal antibody that bound to KDN-gp, but not to KDN-gp depleted of KDN residues. The specificity of the monoclonal antibody, designated mAb.kdn8kdn, was determined by an enzyme-linked immunosorbent assay using KDN-gp samples that varied in KDN content. These antigens were prepared by the selective removal of KDN residues from the native KDN-gp. The mAb.kdn8kdn reacted most strongly with the intact KDN-gp and less strongly with KDN-gp samples containing decreased numbers of KDN residues. The mAb.kdn8kdn was shown specifically to recognize the 28-linked oligo/polyKDN sequences, (8KDN2)
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, and to be able to distinguish specifically (8KDN2)
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chains from (8Neu5Ac2)
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and (8Neu5Gc2)
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chains. The antibody was used successfully for the immunohistochemical detection of reactive KDN epitopes in sections of paraffin embedded rat pancreas. Several controls verified the specificity of the immunohistochemical staining, thus providing the first demonstration of (8KDN2)
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sequences in a mammalian tissue. The mAb.kdn8kdn can now be used to search further for glycoconjugates containing (8KDN2)
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chains and will facilitate studies on their biosynthesis, intracellular localization and function. |
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