Prion diseases are a family of progressive, fatal neurodegenerative disorders caused by the accumulation of the alternatively folded prion protein PrPSc. In the CNS, prions produce neuronal cell death, spongiform vacuolation and gliosis (1). The PrPSc protein is extractable from diseased tissue and biochemically distinguished from endogenous PrPC by partial protease resistance and detergent insolubility (2). Both PrPC and PrPSc share the same amino acid sequence, but PrPSc adopts an abnormal conformation that is transmissible and serves as a template for the conversion of host PrPC into the pathogenic prion isoform (3;4). The mechanism responsible for the transmission, conformational conversion of PrPC to PrPSc, and subsequent disease progression remains enigmatic.
Detection of infectious prions relies on combined use of immunoassay and histopathological assessment of brain tissue from infected animals (5). Current immunoassays are dependant on antibodies that recognize both the normal and abnormal isoforms of PrP. To distinguish abnormal PrPSc from normal PrPC requires limited digestion with proteinase-K (PK) to hydrolyze PK-sensitive PrPC while retaining the PK-resistant PrPSc (PrP 27-30). The PrP 27-30 protein is smaller than PrPC and intact PrPSc and thus can be recognized by a mobility shift following SDS-PAGE and Western blot detection with anti-PrP antibodies (6;7). Yet prion accumulation in the brain is progressive and infected, asymptomatic animals pose significant sampling challenges as minimal accumulation of PrPSc is localized to other more accessible tissue or fluid compartments (8;9). Moreover, variability in the efficacy of prion proteolysis of samples confounds detection of low-level PrPSc (10).
There remains an acute need for a sensitive and selective prion immunodiagnostic assay capable of pre-clinical assessment of infected animals from accessible tissues or fluids (11). Most immunoassay detection limits are insufficient to detect low-level prion contamination that can transmit disease by bioassay. Current assays are confounded by reliance on removal of PK-sensitive PrPC as no antibody has emerged that can selectively distinguish infectious PrPSc from PrPC (12). The need to remove PrPC protein from samples often diminishes immunoassay sensitivity by reducing the amount of PrPSc and increasing assay background. Moreover, the occurrence of PK-sensitive PrPSc isoforms poses additional concerns for many immunodiagnostic assays (13).
The difficulty of prion antibody generation is underscored by the identical primary structure of normal and abnormal PrP protein isoforms and isolation of purified infectious prion. The use of synthetic PrP peptides or recombinant PrPC has been successful in generating anti-PrP antibodies for detection of both PrPC and PrPSc proteins, but use of a PrPC derivative cannot yield an antibody that selectively bind the structurally distinct PrPSc (14;15). Since the primary structure of PrPSc is identical to PrP, a recombinant PrPSc protein cannot be generated. Moreover, the PrPC antigen has proven to be a poor immunogen as endogenous PrPC protein negates a robust immune response (16;17). The immunogenicity of PrPC antigen has been improved by using Prnp-null mice (Prnp0/0) with resulting production of high-affinity anti-PrP antibodies (14). However, the use of a PrPC antigen invariably leads to production of antibodies that recognize PrPC with a low probability of generating a PrPSc selective antibody capable of directly discriminating between normal PrPC and infectious PrPSc.
The most common methods for the diagnostic confirmation of prion disease involve clinical assessment, followed by post-mortem histopathological evaluation of brain tissue along with biochemical detection of PrP 27-30 (21;22). Several problems have confounded the pre-clinical diagnostic detection of prion. First, accumulation of PrPSc increases progressively over time; second, most PrPSc resides in the brain which imposes biopsy challenges. Third, prion concentrations below current immunoassay detection limits can transmit disease in animal bioassay (23;24). Fourth, no direct detection method has been developed that can distinguish PrPSc from PrPC without enzymatic or chemical manipulation to render endogenous PrPC undetectable while retaining PrPSc activity. Indeed, no antibody has emerged that can selectively bind PrPSc but not PrP, moreover, no surrogate analyte has been identified that can identify prions in preclinical animals (22;25). Finally, species and prion strain variability presents additional detection challenges as a result of distinct tissue distribution and availability (26;27).
Useful biochemical methods have emerged for the enrichment of PrPSc from brain homogenates that take advantage of differences in sedimentation and solubility (28;29). Yet, these preparative methods have proven insufficient to yield PrPSc enriched fractions suitable for crystal formation or as immunogen for the generation of PrPSc selective antibodies. Several factors likely contribute to the inability to generate a PrPSc selective antibody. First, the choice and preparation of inoculum have favored the generation of PrPC antibodies. The use of recombinant PrPC invariably yields antibodies that recognize PrP. Moreover, preparation of a native PrPSc is often confounded by contaminating proteins including PrP. Second, wt animals expressing endogenous PrPC may provide a less robust system for the generation of PrPSc antibodies (30). Third, the method used for screening antibodies requires the selective discrimination of those that bind PrPC from those that bind PrPSc. A method that yields that yields abundant PrPSc from diseased tissue and demonstrates a progressive increase in specific infectivity of prions and generation of high-titer antisera with selective activity to PrP is therefore desired.