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 eg gs, and glycoproteins bearing oligo/polymers of deaminated neuraminic acids (KDN) found in the vitelline envelope of the eggs and ovarian fl uid. We report the preparation and characterization of a monoclonal an tibody specifically recognizing oligo/polymers of KDN sequences in gly coproteins and its application in immunohistochemistry. Fusion of sple en cells from a BALB/c mouse immunized with a KDN-rich glycoprotein (K DN-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 antib ody that bound to KDN-gp, but not to KDN-gp depleted of KDN residues. The specificity of the monoclonal antibody, designated mAb.kdn8kdn, wa s determined by an enzyme-linked immunosorbent assay using KDN-gp samp les that varied in KDN content. These antigens were prepared by the se lective removal of KDN residues from the native KDN-gp. The mAb.kdn8kd n reacted most strongly with the intact KDN-gp and less strongly with KDN-gp samples containing decreased numbers of KDN residues. The mAb.k dn8kdn was shown specifically to recognize the alpha 2-->8-linked olig o/polyKDN sequences, (-->8KDN alpha 2-->)(n), and to be able to distin guish specifically (-->8KDN alpha 2-->)(n) chains from (-->8Neu5Ac alp ha 2-->)(n) and (-->8Neu5Gc alpha 2-->)(n) chains. The antibody was us ed successfully for the immunohistochemical detection of reactive KDN epitopes in sections of paraffin embedded rat pancreas. Several contro ls 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 furt her for glycoconjugates containing (-->8KDN alpha 2-->)(n) chains and will facilitate studies on their biosynthesis, intracellular localizat ion and function.