Six mouse monoclonal antibodies have been isolated which react against a bacterial fusion protein containing amino acids 364 to 623 of the NS-1 protein of the prototype strain of the Minute Virus of Mice (MVMp). All six were found to be of the IgG class of antibodies; five being IgG₁ and the sixth being IgG₂[formula omitted]. By immunoblot analyses, these antibodies all recognize an 83 kDa protein found only in MVM-infected mouse fibroblast cells, leading to the assumption that they are all NS-1 specific. Further evidence for this assumption is obtained from indirect immunofluorescence studies showing all but one of the mAbs react against a nuclear protein found in MVM-infected cells.
The epitopes of the antibodies were mapped using carboxy-terminal deleted bacterial fusion proteins derived from the plasmid encoding the original antigen. For the six monoclonal antibodies, four distinct epitopes were found (A - D). Three were clustered in a 16 amino acid region near the carboxy-terminal of the bacterial fusion protein, while the fourth was slightly more toward the amino-terminal side. Competition ELISAs against a 25 amino acid NS-1 specific peptide confirmed the mapping of the A epitope recognized by the CE10 and AC6 monoclonal antibodies.
Also in this thesis, the characterization of a NS-1 fusion protein and a non-fused NS-1 protein expressed in insect cells by recombinant baculoviruses is also described. The latter, a full-length NS-1 protein designated NS-1[formula omitted]ⅽ, was found to be an 84 kDa cytoplasmic protein. This protein was immunoprecipitated by all six monoclonal antibodies. A CE10 monoclonal antibody immunoaffinity column was employed in the single-step purification of NS-1 [formula omitted]c from insect cells. Four elution methods (alkaline, peptide, 6M guanidinium, and acid) were examined and the best purification was obtained using the acid elution. / Medicine, Faculty of / Biochemistry and Molecular Biology, Department of / Graduate
| Identifer | oai:union.ndltd.org:UBC/oai:circle.library.ubc.ca:2429/29405 |
| Date | January 1990 |
| Creators | Yeung, Douglas Edward |
| Publisher | University of British Columbia |
| Source Sets | University of British Columbia |
| Language | English |
| Detected Language | English |
| Type | Text, Thesis/Dissertation |
| Rights | For non-commercial purposes only, such as research, private study and education. Additional conditions apply, see Terms of Use https://open.library.ubc.ca/terms_of_use. |
Page generated in 0.0021 seconds