Generation of monoclonal antibodies against prion proteins with an unconventional nucleic acid-based immunization strategy.
Prion diseases belong to a group of neurodegenerative disorders affecting humans and animals. The human diseases include kuru, Creutzfeldt-Jakob disease (CJD), Gerstmann-Sträussler-Scheinker syndrome (GSS) and fatal familial insomnia (FFI). The pathomechanisms of the prion diseases are not yet understood. Therefore, monoclonal antibodies (mAbs) would provide valuable tools in diagnostics as well as in basic research of these diseases. In contrast to conventional strategies we have developed an immunization protocol based on nucleic acid injection into non tolerant PrP0/0-mice. DNA or RNA coding for different human prion proteins including the mutated sequences associated with CJD, GSS and FFI were injected into muscle tissue. The mice were primarily inoculated with DNA-plasmids encoding PRNP and boosted either with DNA, RNA or recombinant Semliki Forest virus (SFV) particles expressing PRNP. After hybridoma preparation, different mAbs against prion proteins were obtained and their binding behaviour was analysed by peptide-ELISA, Western blot, immunofluorescence and immunoprecipitation. Our mAbs are directed against four different linear epitopes and may also recognize discontinuous regions of the native prion protein. It could, therefore, be demonstrated that immunization of non tolerant mice with DNA and live attenuated SF virus is a valuable means to induce a broad immune response leading eventually to the generation of a panel of mAbs for basic science as well as for diagnostics.
Krasemann
S
S
Laboratory of Molecular Genetic, Mainz, Germany.
Jürgens
T
T
Bodemer
W
W
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000096:biosynthesis*; D001709:Biotechnology; D002460:Cell Line; D004247:DNA / Q000235:genetics / Q000276:immunology; D000939:Epitopes / Q000235:genetics; D005822:Genetic Vectors; D006801:Humans; D007114:Immunization; D051379:Mice; D008969:Molecular Sequence Data; D017096:Prion Diseases / Q000175:diagnosis / Q000276:immunology; D011328:Prions / Q000235:genetics / Q000276:immunology*; D012313:RNA / Q000235:genetics / Q000276:immunology; D012672:Semliki forest virus / Q000235:genetics; D014162:Transfection
D000911:Antibodies, Monoclonal; D000939:Epitopes; D011328:Prions; D012313:RNA; D004247:DNA