Preparation of a novel monoclonal antibody specific for myelin basic protein phosphorylated on Thr98.
Phosphorylation is one of a number of post-translational modifications resulting in charge microheterogeneity of myelin basic protein (MBP). This phosphorylation is claimed to destabilise the compact myelin sheath by decreasing the interaction of membrane bilayers, thereby creating or maintaining pockets of cytoplasm. To further investigate and localise MBP phosphorylation to discrete regions of the myelin sheath we raised a monoclonal antibody with specificity for a known phosphorylation site in MBP. A synthetic peptide was made by Fmoc peptide chemistry and phosphorylation of Thr98 was achieved on the resin by the global phosphorylation methodology, utilising dibenzyl-N,N-diethylphosphoramidite phosphitylation and t-butylhydroperoxide oxidation. The peptide coupled to tuberculin was used to immunise mice for monoclonal antibody production. The selected hybridoma (Clone P12) secreted an IgG2a antibody which reacted strongly with the phosphorylated immunogen and with phosphorylated fractions of bovine MBP obtained by ion exchange chromatography. The antibody had minimal reactivity with the unphosphorylated peptide; the same peptide phosphorylated at another site Ser102; a preparation of unphosphorylated MBP obtained by ion exchange chromatography; and with an irrelevant phosphorylated protein (histone). Similar phosphorylation state-specific monoclonal antibodies could be made to recognise other specific phosphorylation sites in MBP or other proteins. It is planned to use these antibodies to quantify and locate the extent of MBP phosphorylation in normal and multiple sclerosis myelin.
Yon
M
M
School of Biological and Molecular Sciences, Oxford Brookes University, Headington, UK.
White
P
P
Groome
N
N
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000502:physiology*; D051379:Mice; D008807:Mice, Inbred BALB C; D008969:Molecular Sequence Data; D009185:Myelin Proteins / Q000502:physiology*; D010766:Phosphorylation
D000911:Antibodies, Monoclonal; D009185:Myelin Proteins