Related Experiment Videos
Preparation of a novel monoclonal antibody specific for myelin basic protein phosphorylated on Thr98
1School of Biological and Molecular Sciences, Oxford Brookes University, Headington, UK.
Abstract:
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.
Insights
Researchers developed a novel monoclonal antibody to specifically detect phosphorylated myelin basic protein (MBP). This tool will help quantify and locate MBP phosphorylation in normal and multiple sclerosis myelin.
Area of Science:
- Neuroscience
- Biochemistry
- Immunology
Background:
- Myelin basic protein (MBP) undergoes post-translational modifications, including phosphorylation, leading to charge microheterogeneity.
- MBP phosphorylation is hypothesized to destabilize the myelin sheath, potentially impacting its structure and function.
- Understanding the localization and extent of MBP phosphorylation is crucial for myelin research, particularly in neurological diseases.
Purpose of the Study:
- To develop a highly specific monoclonal antibody for a known phosphorylation site on myelin basic protein (MBP).
- To investigate and localize MBP phosphorylation within discrete regions of the myelin sheath.
- To create a tool for quantifying and mapping MBP phosphorylation in both normal and multiple sclerosis (MS) myelin.
Main Methods:
- Synthesized a phosphopeptide representing a specific phosphorylation site (Thr98) on MBP using Fmoc peptide chemistry and on-resin phosphorylation.
- Immunized mice with the phosphorylated peptide coupled to tuberculin to generate hybridomas.
- Screened hybridomas to select a clone (P12) producing an IgG2a antibody with specificity for phosphorylated MBP.
Main Results:
- The P12 monoclonal antibody demonstrated strong reactivity with the phosphorylated immunogen and phosphorylated bovine MBP fractions.
- The antibody exhibited minimal cross-reactivity with unphosphorylated MBP, MBP phosphorylated at a different site (Ser102), or irrelevant phosphorylated proteins.
- This indicates high specificity for the targeted phosphorylation site on MBP.
Conclusions:
- A phosphorylation state-specific monoclonal antibody (Clone P12) was successfully generated for myelin basic protein (MBP).
- This antibody can accurately detect and potentially localize specific phosphorylation events within MBP.
- The developed antibody represents a valuable tool for future studies on MBP phosphorylation in neurological conditions like multiple sclerosis.