Date Available

6-6-2011

Year of Publication

2011

Degree Name

Master of Science (MS)

Document Type

Thesis

College

Medicine

Department

Microbiology

First Advisor

Dr. Glenn Telling

Abstract

Monoclonal antibodies (mAbs) recognizing different regions of PrP are potential tools in the study of prion diseases and immunotherapy. We used shuffled recombinant prion protein containing elk and mouse PrP as antigen to produce monoclonal antibodies in mice. We found that mAb 5C6 mapped to a discontinuous epitope comprised of amino acid 132 and 158 (mouse numbering). Monoclonal anibody 9E9 which maps to a unique N-terminal epitope at amino acid preferentially recognized cervid PrP. In contrast, the epitope of mAb 9H9 is located in the C-terminus and only reacted with mouse and hamster. The epitope for mAb 7H11 appears to be affected by the glycosylation of PrP and by the presence or absence of the disulfide bond. To confirm the epitopes of these mAbs, we constructed elk and mouse mutants both with and without reactivity to 5C6 and 9E9. We then used these mutants to investigate the effect of each epitope on the conversion of PrPC to PrPsc. In one approach to map the epitopes of newly-generated monoclonal antibodies (mAbs), we generated a series of contiguous ten amino acids deletion constructs spanning amino acids 107 to 230 and expressed these recombinant proteins in mammalian cells (RK13) or bacteria. Using Western blotting, all deletion constructs could be recognized with antibodies to the extreme C-terminus of PrP, or the N-terminal region upstream of the structured globular domain of PrP. However, mAb 5C6 failed to react with all internally deleted PrP constructs expressed in mammalian cells, and to a lesser extent bacterially produced mutant recombinant proteins. We confirmed the surprising result using the well-defined antibodies 6H4 and D18, which recognize epitopes in the same internal region as 5C6. Our results suggest the formation of an ultra-stable, SDS-resistant conformation in PrP harboring deletions mutations in the globular domain of PrP. We hypothesize that epitope burying within this stable conformation(s) precludes mAb recognition by 5C6, 6H4 and D18. It will be of extreme interest to determine the relationship of this previously undefined PrP conformation to the pathogenic process of PrP conformational change.

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