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Previously Funded
National Prion Research Highlights

2006:



2006

Detection of Prion Aggregates
Posted December 11, 2006
Man-Sun Sy, Ph.D., Case Western Reserve University School of Medicine, Cleveland, Ohio

Prion diseases, or transmissible spongiform encephalopathies (TSEs), are caused by the conversion of the endogenously occurring prion protein (PrPC) to the disease-causing prion protein conformation (PrPSc). It is thought that this conversion occurs as a result of interaction between PrPC and PrPSc, which serves as a template to induce conformational change in PrPC from a predominately alpha-helical conformation to a predominately beta-sheet structure. This change is associated with the carboxy-terminal of PrPSc becoming protease-resistant. A minor, yet critical, difference exists between normal and disease-associated conformers; thus, discrimination between the forms is difficult, making detection of prion disease problematic. Currently, diagnostic tests for prion disease rely heavily on demonstration of protease-resistant forms of prion protein or the uncovering of hidden epitopes. In a Fiscal Year 2002 Department of Defense National Prion Research Program Investigator-Initiated Research Award, Dr. Man-Sun Sy proposed to develop an improved enzyme-linked immunosorbent assay (ELISA) for the diagnosis of TSEs. Because prion proteins dimerize, or form aggregates, Dr. Sy reasoned that certain epitopes would become inaccessible to antibody-binding, while concurrently presenting other epitopes in multiples, depending upon the composition of the aggregate. Dr. Sy concluded that if the epitope were present more than once, the same monoclonal antibody could be used as both capture and detection antibodies in the ELISA. After confirming the presence of dimers in recombinant human, mouse, ovid, and bovine prion protein preparations, 30 anti-PrP monoclonal antibodies were screened for their ability to recognize recombinant mouse PrP dimers (rMo-PrP). Five antibodies, four of which also recognized recombinant dimers from the other species, were found to react strongly, suggesting common features in their central regions, where the antibodies bind. An ELISA employing these four antibodies revealed that the four reacted strongly and with specificity against rMo-PrP dimers. Another ELISA found four antibodies that strongly recognized PrP in brain homogenates from infected mice. Only one antibody, 11G5, whose epitope is amino acids 114-130, was positive in both ELISAs, suggesting differences between in vitro and in vivo dimers. Additionally, it was found that the ELISA worked for three differing PrP strains and predominately recognized PrPSc aggregates of varying sizes from infected mice. The enhanced sensitivity was exemplified by detection of PrPSc 70 days post-infection, whereas previous assays required an incubation time of 140 days. Dr. Sy extrapolates that the ELISA has the potential to detect between 0.06 µg and 0.006 µg of aggregated PrP. ELISA results also imply a change in the helix 1 region of PrP through conversion of PrPC to PrPSc and possible interaction between the amino and carboxy-terminals of recombinant human PrP (1). Dr. Sy and colleagues have further refined the assay, combining an in vitro amplification step, the aggregation-specific ELISA, and a fluorescent amplification catalyzed by T7 RNA polymerase. The resulting assay, termed Am-A-FACCT, was found to have 100% specificity and sensitivity in detecting disease-associated prion aggregates in blood of infected mice at a late, but asymptomatic, stage. Furthermore, Am-A-FACCT was shown to have 50% sensitivity and 100% specificity at a very early time point following infection. Importantly, the assay was able to detect prion aggregates in the blood of mule deer with the naturally occurring prion disease, chronic wasting disease, implicating its potential use in safeguarding the food supply and preventing human contagion (2).

Publications:

Pan T, Chang B, Wong P, et al. 2005. An aggregation-specific enzyme-linked immunosorbent assay: Detection of conformational differences between recombinant PrP protein dimers and PrPSc aggregates. Journal of Virology 79(19):12355-12364.

Chang B, Cheng X, Yin S, et al. 2006. A blood test for prion: Disease associated prion aggregate is detected in the blood of infected but asymptomatic animals. Clinical Vaccine Immunology 2006 Nov 1; [Epub ahead of print].

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