MONOCLONAL-ANTIBODY SPECIFIC FOR ALPHA-2-]8-LINKED OLIGO DEAMINATED NEURAMINIC ACID (KDN) SEQUENCES IN GLYCOPROTEINS - PREPARATION AND CHARACTERIZATION OF A MONOCLONAL-ANTIBODY AND ITS APPLICATION IN IMMUNOHISTOCHEMISTRY

Two particular types of sialoglycoproteins have been detected in fish: polysialoglycoproteins containing alpha 2-->8-linked polysialic acid (-->8Neu5Gc alpha 2-->)(n), 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 (-->8KDN alpha 2-->)(n)-->6(KDN alpha 2-->3Gal beta 1-->3G alpha 1NA-c alpha 1-->3) GalNAc alpha 1--> 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 alpha 2-->8-linked oligo/polyKDN sequences, (-->8KDN alpha 2-->)(n), and to be able to distinguish specifically (-->8KDN alpha 2-->)(n) chains from (-->8Neu5Ac alpha 2-->)(n) and (-->8Neu5Gc alpha 2-->)(n) 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 (-->8KDN alpha 2-->)(n) sequences in a mammalian tissue. The mAb.kdn8kdn can now be used to search further for glycoconjugates containing (-->8KDN alpha 2-->)(n) chains and will facilitate studies on their biosynthesis, intracellular localization and function.