Monoclonal antibodies to oligodendrocytes: Characterization and developmental profiles

Abstract

During the last decade considerable attention has been focused on delineating antigenic markers that can be used to dissect complex interactions at the oligodendrocyte cell surface. Several markers are known at the present time, although most of these are directed to known myelin components. We have used bulk-isolated bovine oligodendrocytes maintained in culture as immunogen, in the hope of generating antibodies to previously unidentified oligodendrocyte-specific surface molecules. Immunizations in vivo and in vitro followed by standard hybridoma protocols, generated three monoclonal antibodies, E9, 75 and 120. The cellular and tissue distribution of the antibodies and the characterization of the putative antigens they recognize has been carried out by biochemical and immunocytochemical analyses.;By indirect immunofluorescence, all three antibodies stained bovine oligodendrocytes in culture, and mAbs E9 and 75 stained rat oligodendrocytes. In order to determine the cellular specificities of mAbs E9, 75 and 120, primary cultures and cell lines were tested for immunoreactivity. MAb 75 bound to a population of bipotential progenitor cells in rat forebrain cultures, to Schwann cells and to several mouse, rat and human tumor lines. MAb E9 recognized an antigen expressed on galactocerebroside-positive cells in dissociated rat cultures.;Further characterization of the antigens was carried out by light microscopy and electron microscopy on tissue sections. Localization of mAbs E9, 75 and 120 was to the outer lamellae of myelinated fibers and to the plasma membrane of oligodendrocytes.;In order to identify the molecular nature of these antigens, standard biochemical procedures for lipid and protein analyses were performed. Analysis of lipids by ELISA demonstrated that mAbs E9 and 75 bound to total lipid extracts from bovine white matter and myelin. Further examination by TLC immunoblots of ganglioside extracts from white matter indicated that mAb 75 was able to detect an antigen that migrated above ganglioside GM4. These results suggested that mAb E9 may recognize a lipid antigen and mAb 75 may be directed to a complex glycolipid. The sensitivity of the antigen identified by mAb 120 to digestion by trypsin indicated involvement of a protein moiety.