New method for detection of prion diseases

描述(申请人提供)：我们提出了基于多光子检测(MPD)[1，2，3，4]技术与蛋白质组学[5，6，7，8]相结合的发现TSE生物标记物的新方法，特别是通过应用MPD增强蛋白质差异显示(dd-Prot/MPD)和免疫分析(IA/MPD)。随后，该方法将扩展到MPD增强型超敏蛋白芯片(P-CHIP/MPD)。这些由A.K.Drukier博士团队开发的MPD增强型技术，使低丰度蛋白(“分子开关”)水平与TSE感染之间的关联能够进行更灵敏、更便宜的测试。这些差异显示的蛋白质随后将被确认为TSE生物标记物。 这项建议的第一阶段的主要目标是发现在TSE病例中下调的差异显示蛋白。几年前，M.哈林顿博士对CJD患者进行了一项类似的研究。这项开创性的研究是使用银染2D凝胶进行的，并揭示了那些差异显示的蛋白质在脑脊液中存在相对较高的水平(1-10fmole/ml)。只发现了一个明显上调的蛋白质和几个可能下调的蛋白质。蛋白质组学的新工具，如dd-prot/mpd和高灵敏质谱学，可能允许在少数阿托摩尔/毫升水平上进行检测和鉴定。我们的研究使用对阿托莫尔/毫升水平敏感的蛋白质组学方法，有望检测到数十个差异表达的蛋白质。希望其中一些也会出现在血液中，以便在死前进行TSE诊断。 通过基因组信息鉴定蛋白质，可以合成重组蛋白质和开发合适的单抗。如果几个TSE生物标志物的重要性得到确认，我们将开发专门的超灵敏P-芯片(P-CHIP/MPD)，用于定量高达50fg/ml的靶向差异显示蛋白，从而开发出低成本的死前TSE检测方法。

DESCRIPTION (provided by applicant): 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.