We propose new methods for discovery of TSE biomarkers based on combing the MultiPhoton Detection (MPD) [1,2,3,4] technique with methods of proteomics [5,6,7,8], especially by applying MPD enhanced differential display of proteins (dd-PROT/MPD) and immunoassay (IA/MPD). Subsequently, the methods will be extended to MPD enhanced supersensitive protein chips (P-chips/MPD). These MPD enhanced techniques, developed by the group of Dr. A. K. Drukier, enable more sensitive and less expensive tests for correlating the levels of low abundance protein ("molecular switch") with TSE infections. These differentially displayed proteins will then be validated as TSE biomarkers. The primary objective of Phase I of this proposal is to discover differentially displayed proteins that are down regulated in TSE cases. A similar study was conducted a few years ago by Dr. M. Harrington, on CJD patients. This pioneering study was performed using silver stained 2D gels and revealed those differentially displayed proteins present at relatively high levels (1-10 fmole/ml) in CSF. Only one clearly up-regulated protein and a few possibly down-regulated proteins were discovered. The new tools of proteomics, such as dd-PROT/MPD and high-sensitivity mass spectroscopy, may allow detection and identification at the few attomole/ml level. Our study using proteomics methods sensitive to attomole/ml levels is expected to detect a few tens of differentially expressed proteins. Hopefully, some of them will also be present in blood to allow ante mortem TSE diagnosis. The identification of proteins via genomic information permits the synthesis of recombinant proteins and development of appropriate monoclonal antibodies (mAbs). If the importance of several TSE biomarkers is confirmed, we will develop dedicated supersensitive P-chips (P-chips/MPD) for quantifying up to 64 targeted differentially displayed proteins at 50 fg/ml. Thus, low cost ante mortem TSE detection methods will be developed.