This project aims to optimise the process conditions for five candidate strain-typing tests (Test A-E),and characterise and optimise the PAGE/Western blotting procedures,following protease digestion of samples. Additionally,application of immunoaffinity precipitation of PrPsc to strain typing assays and analysis of bovine BSE, ovine scrapie and ovine BSE samples using the strain-typing assays will be performed.
To determine whether the five protease tests developed in project SE1766 could be useful tools for strain-typing, they will be optimised and standardised. Optimisation will be achieved by evaluation of the effects of pH, buffer composition, enzyme kinetics, and antibody probes.To increase the specificity and sensitivity of the strain-typing assays, de-glycosylation and high-resolution analysis of the samples will be carried out.
Firstly, de-glycosylation of PrPSc will be optimised after protease digestion and samples analysed in the strain-typing assays. The de-glycosylated protein fragments will be separated by 2D IEF/SDS-PAGE electrophoresis. The antibodies used in the strain-typing assays have so far been primarily 6H4, SAF32 and P4, but FH11 and AG8 could also be useful differentiating probes. The antibody epitopes will be mapped using phage-display peptide libraries to provide further explanation of the efficacy of these assays and allow more detailed identification of the PrP fragments produced.
Studies will be carried out to determine whether treatment of enriched PrPres fractions by immunoprecipitation, using the IDEXX monoclonal antibodies, raised to the YYR epitope, prior to protease treatment could increase test sensitivity and reproducibility.The refined strain-typing assays will be assessed and applied to the known, available strains of ovine scrapie and BSE from bovine brains collected pre-1996 and post-1998. In addition, samples of CNS tissue from 'unclassified' scrapie cases will be evaluated, as well as ovine BSE samples.