Monoclonal antibodies against myelin proteolipid protein: identification and characterization of two major determinants.
This report describes the preparation and characterization of a panel of monoclonal antibodies (mAbs) against the myelin proteolipid protein (PLP). A Lewis rat was immunized with bovine proteolipid apoprotein and 27 mAbs were selected based on their reactivity against bovine PLP on enzyme-linked immunosorbent assays. Eleven mAbs recognized the PLP carboxyl-terminal sequence when tested against a panel of synthetic peptides in a solid-phase assay. A carboxyl-terminal pentapeptide (residues 272-276) was sufficient for antibody binding and the terminal phenylalanine residue was found particularly important. Deletion, modification, or replacement of this residue markedly reduced or obliterated antigen-antibody interaction. Nine mAbs reacted with a second antigenic determinant, residues 209-217, but these could be identified only by competitive immunoassays. This peptide was a more effective inhibitor than the longer peptides 202-217 and 205-221, suggesting that flanking residues may interfere with peptide-antibody interaction. Seven antibodies did not react with any of the synthetic peptides tested and their determinants remain unidentified. Immunoblot analysis showed that the mAbs reacted with both the PLP and the DM-20 isoforms. Twenty-three of the mAbs were of the immunoglobulin G2a or b isotype; the remaining antibodies were immunoglobulin M and all of these were specific for residues 209-217. Cultured murine oligodendrocytes were stained by most of the mAbs tested, but the most intense reactivity was observed with the carboxyl-terminus-specific mAbs. The immunocytochemical analyses demonstrate that the mAbs react with the native PLP in situ and show their potential usefulness for studies of the cell biology of myelin and oligodendrocytes.
Yamamura
T
T
Biochemistry Division, E.K. Shriver Center, Waltham, MA 02254.
Konola
J T
JT
Wekerle
H
H
Lees
M B
MB
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal; D000918:Antibody Specificity; D001921:Brain / Q000166:cytology; D002417:Cattle; D002478:Cells, Cultured; D004797:Enzyme-Linked Immunosorbent Assay; D000939:Epitopes / Q000032:analysis*; D005455:Fluorescent Antibody Technique; D015151:Immunoblotting; D007074:Immunoglobulin G / Q000145:classification; D008969:Molecular Sequence Data; D009185:Myelin Proteins / Q000032:analysis* / Q000276:immunology; D018991:Myelin Proteolipid Protein; D009836:Oligodendroglia / Q000166:cytology*; D010455:Peptides / Q000138:chemical synthesis / Q000276:immunology; D051381:Rats; D011917:Rats, Inbred Lew / Q000276:immunology
D000911:Antibodies, Monoclonal; D000939:Epitopes; D007074:Immunoglobulin G; D009185:Myelin Proteins; D018991:Myelin Proteolipid Protein; D010455:Peptides