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Epitope mapping of monoclonal antibodies raised to recombinant Mengo 3D polymerase
1Institute for Molecular Virology, University of Wisconsin-Madison 53706, USA.
Abstract:
The cDNA coding sequence of the RNA-dependent RNA polymerase (3Dpol) of Mengovirus was cloned and expressed in a bacterial system. Eleven monoclonal antibodies were raised against the recombinant Mengo 3Dpol (rM3D). All of them recognized the recombinant and the viral-induced form of the protein. The panel of monoclonals belonged to the IgG1 and IgG2a isotypes and were mapped to four different epitopes in the 3D molecule by competition assays. All monoclonals recognized Mengo 3Dpol in western blots and cross-reacted with the homologous polymerases of seven other cardioviruses but failed to react with 3Dpol from poliovirus type 1 and 3 or rhinovirus type 14 and 16.
Insights
Researchers developed monoclonal antibodies against Mengovirus RNA-dependent RNA polymerase (3Dpol). These antibodies recognize both recombinant and viral 3Dpol, cross-reacting with cardioviruses but not polioviruses or rhinoviruses.
Area of Science:
- Virology
- Molecular Biology
- Immunology
Background:
- RNA-dependent RNA polymerase (3Dpol) is crucial for viral replication.
- Mengo virus 3Dpol is a key target for antiviral strategies.
- Characterizing viral proteins aids in understanding viral pathogenesis and developing diagnostics.
Purpose of the Study:
- To generate and characterize monoclonal antibodies against Mengovirus 3Dpol.
- To investigate the antigenic relationship between Mengo 3Dpol and other viral polymerases.
- To identify specific epitopes on the Mengo 3Dpol molecule.
Main Methods:
- Cloning and bacterial expression of Mengovirus 3Dpol.
- Generation of monoclonal antibodies (mAbs) against recombinant 3Dpol (rM3D).
- Epitope mapping using antibody competition assays and Western blotting.
Main Results:
- Eleven mAbs were successfully raised against rM3D, recognizing both recombinant and viral forms.
- The mAbs belonged to IgG1 and IgG2a isotypes and recognized four distinct epitopes.
- All mAbs cross-reacted with 3Dpol from seven other cardioviruses but showed no reactivity with poliovirus or rhinovirus 3Dpol.
Conclusions:
- The generated mAbs are specific tools for studying Mengovirus 3Dpol.
- These antibodies confirm conserved epitopes within cardioviruses.
- The lack of cross-reactivity with poliovirus and rhinovirus highlights antigenic differences between picornaviruses.