Screening for pathogens has been an essential element in the defense against blood-borne diseases. The most likely target for a diagnostic for CJD is the intrinsically insoluble, protease-resistant, infection-specific amyloid, PrPres, that is closely associated with infectivity in most, but not all, contexts. Detection and analysis of PrPres has been by Western blot or other immunoassays with maximum sensitivities equivalent to 103 to 104 infectious units of TSE. In contrast, the blood of clinically affected rodents contains from 5 to 25 iu/ml. Titers may be even lower earlier in the disease. As a consequence, PrPres has not, as yet, been demonstrated in blood. Moreover, a significant proportion of blood-borne infectivity seems to be more soluble than would be expected for brain-derived infectivity, suggesting that it may not be associated with PrPres. Before we can sensibly interpret the assay of PrPres in human bloods of unknown (and unknowable) infectivity, we must first validate our ability to detect PrPres in a blood of known infectivity. Therefore our first objective will be to demonstrate proof of principal for a PrPres based diagnostic by using whatever means necessary to detect PrPres in blood and/or plasma known to contain TSE infectivity as verified by direct measurement in rodent assays. We will extend our current work using the animal infectivity assay to identify cellular and plasma fractionations that concentrate infectivity and are thereby most likely to concentrate the PrPres as well. We will then focus our attempts to demonstrate PrPres on those fractions containing the highest concentrations of infectivity. By concentrating PrPres, 100 to 1000 fold from intrinsically infected hamster blood or plasma, prepared using proven methods that we have developed, we should be able to bring the PrPres signal into range of the currently available immunoassays. A successful first demonstration of PrPres in blood will likely require methods that are not directly applicable to mass screening protocols, but will nevertheless identify appropriate targets for refinement into more efficient assays. Having established our ability to detect PrPres in blood, we will focus further efforts on simplifying the detection with the goal of developing a method more suitable for mass screening. These methods and refinements should be applicable to any detection system. We will use lanthanide-based time resolved fluorescence to achieve maximum sensitivity in a mass-screening ELISA format. However, our expectation is that, working at the limits of detection, all positives will have to be confirmed by the more definitive Western blot assay. Therefore we will also continue our development of a highly sensitive and quantitative Western blot method based on antigen capture and quantitative assay by fluorimaging. As each of our objectives are achieved, we will apply the methods to detection of PrPres in mouse adapted nv-CJD and to blood from human CJD patients.
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