Production and characterization of monoclonal antibodies to the extracellular domain of P0.
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 human P0-ED were selected by ELISA and were characterized by Western blot, immunohistochemistry, and a competition assay. Antibodies belonged to the IgG or IgM class, and P04-P07, reacted with P0 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 enzyme-linked 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 P0 during myelination and in demyelinating diseases of the peripheral nervous system.
Archelos
J J
JJ
Department of Neurology, Julius-Maximilians-Universität Würzburg, Germany.
Roggenbuck
K
K
Schneider-Schaulies
J
J
Linington
C
C
Toyka
K V
KV
Hartung
H P
HP
D000818:Animals; D000911:Antibodies, Monoclonal; D000918:Antibody Specificity; D001921:Brain / Q000166:cytology; D015816:Cell Adhesion Molecules, Neuronal / Q000032:analysis*; D004797:Enzyme-Linked Immunosorbent Assay; D005260:Female; D006801:Humans; D015151:Immunoblotting; D007150:Immunohistochemistry; D051379:Mice; D008807:Mice, Inbred BALB C / Q000276:immunology; D008970:Molecular Weight; D018993:Myelin P0 Protein; D009185:Myelin Proteins / Q000032:analysis* / Q000276:immunology; D009186:Myelin Sheath / Q000737:chemistry; D010455:Peptides / Q000138:chemical synthesis / Q000276:immunology; D010525:Peripheral Nerves / Q000737:chemistry; D051381:Rats; D011994:Recombinant Proteins / Q000032:analysis / Q000276:immunology; D012584:Sciatic Nerve / Q000166:cytology
D000911:Antibodies, Monoclonal; D015816:Cell Adhesion Molecules, Neuronal; D018993:Myelin P0 Protein; D009185:Myelin Proteins; D010455:Peptides; D011994:Recombinant Proteins