Novel Reagent Development for Chronic Wasting Disease

Mice and chickens will be immunized with peptide conjugates and fusions with appropriate myeloma fusion partners will be performed when serum specificity is ascertained. Isolated hybridoma clones specific for cyclized peptides but not un-cyclized peptide by solid phase ELISA will be isolated and expanded. Purified and enzyme-conjugated monoclonal antibody reagents will be made and validation of the potential utility of these antibodies will be assessed by specificity for PrPres, but not the PrPsen.

Chronic wasting disease (CWD) in elk and deer, and scrapie in sheep are a family of degenerative, central nervous system diseases known as transmissible spongiform encephalopathies (TSEs) or prion diseases. They are often called spongiform because of the post mortem appearance of hole-like structures called vacuoles in the cortex and cerebellum of the brain. Chronic wasting disease is the result of the accumulation in the brain of a normal cell surface-expressed protein, called PrP or PrPsen, that has re-folded (fromnalpha-helical to beta-sheet) and aggregated into an entity, PrPres, that interrupts normal neuronal activity. The accumulation of PrPres is considered to be directly responsible for the onset of the pathology observed with CWD and is also considered the main cause of other TSEs. To date, no suitable antibody reagents are available that can distinguish between the normal and the pathological form of the prion protein in CWD, and there is urgency and a significant requirement in the marketplace for reagents of this nature. Current tests for CWD are lengthy, involve extensive pre-treatment and are only available as "dead animal" tests. An opportunity exists for New England Rare Reagents to produce specific antibodies to CWD PrPres. The benefit from such a reagent will allow for more specific and less labor-intensive testing to be performed. These improvements should make deer and elk testing more available to state facilities attempting to control and eventually eradicate the disease.

To generate synthetic immunogens with beta-sheet associations, peptides will be cyclized on resin by de-protecting strategically inserted cysteine residues to generate intra-molecular disulfide bridges. These peptides, subsequently cleaved from the resin, are then chemically conjugated with T-cell epitope peptides. These conjugates should overcome any tolerance to self-proteins and generate useful IgG responses to the self-peptides. Both mice and chickens will be immunized because the chicken is phylogenetically removed from the mouse and the potential exists whereby the immunological repertoire of the chicken is unique enough and able, in this case, to generate a specificity that the mouse cannot and may, therefore, recognize unique structures in the universe. Antibody screening will be focused toward an ability to preferentially recognize cyclized peptides and not the linear counterparts, but utility for further analysis will rest upon the ability of the antibodies to recognize the actual re-folded prion in diseased tissue. Dr. French will utilize purified and conjugated monoclonal reagents to undertake these studies by undertaking immuno-precipitation, dot blot assay, western blot analysis, immunohistochemistry and immuno-electron microscopy of both cervid and mouse scrapie-model brain tissue isolates.