PRODUCTION AND CHARACTERIZATION OF MONOCLONAL-ANTIBODIES TO THE EXTRACELLULAR DOMAIN OF PO

Seven monoclonal antibodies were raised against the immunoglobulin-lik e extracellular domain of (P0-ED), the major protein of peripheral ner vous system myelin. Mice were immunized with purified recombinant rat P0-ED. After fusion, 7 clones (P01-P07) recognizing either recombinant , rat, mouse, or human P0-ED were selected by ELISA and were character ized by Western blot, immunohistochemistry, and a competition assay. A ntibodies belonged to the IgG or IgM class, and P04-P07, reacted with P0 in fresh-frozen and paraffin-embedded sections of human or rat peri pheral nerve, but not with myelin proteins of the central nervous syst em of either species. Epitope specificity of the antibodies was determ ined by a competition enzyme-linked immunosorbent assay (ELISA) and a direct ELISA using short synthetic peptides spanning the entire extrac ellular domain of P0. These assays showed that P01 and P02 exhibiting the same reaction pattern in Western blot and immunohistochemistry rea cted with different distant epitopes of P0. Furthermore, the monoclona l antibodies P05 and P06 recognized 2 different epitopes in close prox imity within the neuritogenic extracellular sequence of P0. This panel of monoclonal antibodies, each binding to a different epitope of the extracellular domain of P0, will be useful for in vitro and in vivo st udies designed to explore the role of P0 during myelination and in dem yelinating diseases of the peripheral nervous system.