The use of peptide-mediated electrofusion to select monoclonal antibodies directed against specific and homologous regions of the potyvirus coat protein.
Whilst monoclonal antibodies (Mab) to potyviruses have been generated, it has not been possible to produce molecules with high specificity or broad reactivity to defined conserved amino acid sequences. In the current study, peptide-mediated electrofusion was used to select for high efficiency antibody-secreting hybridomas after mice were immunized with highly immunogenic viral coat protein. Mice were immunized with coat protein from either one potyvirus (potato virus Y, PVY-D) or a mixture of five distinct potyviruses. Two well-defined peptides were used for selective electrofusions. Peptide-1 was selected from the highly specific N terminal region of PVY-D and peptide-2 from the highly conserved N terminal/core junction region of Johnson grass mosaic virus (JGMV). Conventional PEG-mediated fusions using mice immunized with these peptides did not result in hybridoma formation. On the other hand, electrofusions using biotin-streptavidin to bridge peptide-specific B cells to myeloma cells produced hybridomas secreting antibodies either highly specific to PVY-D or cross-reactive with all potyviruses, depending on the peptide used.
Werkmeister
J A
JA
CSIRO, Division of Biomolecular Engineering, Parkville, Victoria, Australia.
Tebb
T A
TA
Kirkpatrick
A
A
Shukla
D D
DD
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000096:biosynthesis / Q000276:immunology*; D000918:Antibody Specificity / Q000276:immunology; D002213:Capsid / Q000276:immunology*; D002459:Cell Fusion; D004797:Enzyme-Linked Immunosorbent Assay; D000939:Epitopes / Q000276:immunology; D005260:Female; D006825:Hybridomas / Q000276:immunology; D007158:Immunologic Techniques; D051379:Mice; D008807:Mice, Inbred BALB C; D008969:Molecular Sequence Data; D009029:Mosaic Viruses / Q000276:immunology*; D010942:Plant Viruses / Q000276:immunology*; D011198:Solanum tuberosum; D014764:Viral Proteins / Q000276:immunology*
D000911:Antibodies, Monoclonal; D000939:Epitopes; D014764:Viral Proteins