Production and characterization of monoclonal antibodies to the extracellular domain of P0

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Abstract
Seven monoclonal antibodies were raised against the immunoglobulin-like extracellular domain of P0 (P0-ED), the major protein of peripheral nervous system myelin. Mice were immunized with purified recombinant rat P0-ED. After fusion, 7 clones (P01–P07) recognizing either recombinant, rat, mouse, or hu-man P0-ED were selected by ELISA and were characterized by Western blot, immunohistochemistry, and a competition assay. Antibodies belonged to the lgG or lgM class, and P04–P07, reacted with PO in fresh-frozen and paraffin-embedded sections of human or rat peripheral nerve, but not with myelin proteins of the central nervous system of either species. Epitope specificity of the antibodies was determined by a competition enzymelinked immunosorbent assay (ELISA) and a direct ELISA using short synthetic peptides spanning the entire extracellular domain of P0. These assays showed that P01 and P02 exhibiting the same reaction pattern in Western blot and immunohistochemistry reacted with different distant epitopes of P0. Furthermore, the monoclonal antibodies P05 and P06 recognized 2 different epitopes in close proximity 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 studies designed to explore the role of PO during myelination and in demyelinating dis-eases of the peripheral nervous system.