Searching for Reliable Premortem Protein Biomarkers for Prion Diseases

Progress and Challenges to Date

Di Ma; Lingjun Li


Expert Rev Proteomics. 2012;9(3):267-280. 

In This Article

PrPSc as a Biomarker for Prion Disease Diagnosis

Given that there is no cure for prion diseases, it is imperative to develop effective diagnostic assays for routine screening in large populations to prevent a potential spread of the disease from animal to human or from human to human. Currently, all commercial diagnostic tests for TSEs are carried out by the direct detection of PrPSc or PrPC in brain tissue. Conventionally, histological and immunohistological methods have served as reference for clinical diagnosis by detection of characteristic vacuolar or spongiform changes in specific areas of the brain.[18] The recent BSE epidemic has led to the development of antibody-based commercial diagnostic assays for BSE, scrapie and CWD. Antibody-based assays utilize a Western blot/ELISA approach to provide more rapid and large-scale analysis, which is now a routine TSE diagnostic assay for use in animals.[19–21] Recently, the capability of a mass spectrometry (MS)-based method for more sensitive detection and quantification of PrPSc has been exploited. Carter's group reported a MS-based multiple reaction monitoring (MRM) method that can be used to both confirm and quantitate PrP 27–30 derived trypsin fragments in several different species.[22–24] The limit of detection of this method (20–30 amol) is considerably lower than that of any that has been previously described.

Although sensitive and specific, all current commercial diagnostic kits are supplied only for animal TSE diseases and can only be applied to postmortem diagnosis for discrimination and confirmation of the presence of PrPSc. Ugnon-Café et al. reported that two commercial assays developed based on ELISA and Western blot can be used to detect PrPSc in cerebral and lymphoid tissues of TSE patients.[25] It was shown that both tests were specific and quite sensitive in the detection of PrPSc in postmortem brains of affected patients but not in the controls. Furthermore, two premortem tonsils from three patients with vCJD were detected correctly, suggesting that these assays could be used for human TSE diagnosis, and that detection of PrPSc in the tonsil could be used as premortem diagnosis for CJD. Due to the fact that the concentration of PrPSc in peripheral tissue is substantially lower than that in neural tissues, the sensitivity of conventional immunoassays is not high enough for the detection of PrPSc in easily accessible body fluids for preclinical diagnosis. However, when coupled with an enrichment step of PrPSc from biological samples, the sensitivity of immunodetection could be significantly increased. Edgeworth et al. developed a quantitative blood-based assay for vCJD diagnosis where they used a solid-state binding matrix to capture and concentrate PrPSc from whole blood of vCJD patients and coupled this method to direct immunodetection of surface-bound material.[26,27] A masked panel of 190 whole blood samples from normal controls and patients with vCJD, sCJD and other neurological diseases were analyzed by this assay, and it showed a sensitivity of 71.4% and a specificity of 100%. Although the sensitivity of this assay is yet to be improved, the findings from this study demonstrated the feasibility of preclinical diagnosis of vCJD by blood assay. Recently, protein misfolding cyclic amplification (PMCA) technology, which was developed in 2001 by Claudio Soto's group,[28] has been applied to the detection of PrPSc with enhanced sensitivity.[29] PMCA is based on the ability of a small amount of PrPSc to convert an excess amount of normal PrPC to a proteinase K-resistant form to the level that can be detected by conventional immunoassays, thus 'amplifying' the original infectious seed. Amplification can be increased by breaking down the growing chain of PrPSc to smaller units, which, in turn, act as seeds for further replication. PMCA enabled the development of ultrasensitive tests for prions and prion diseases. The PMCA has been reported for premortem detection of CWD infection in the peripheral tissues of cervids.[30] However, the use of PrPSc as a preclinical biomarker for surveillance will be challenging due to the fact that the amount of PrPSc is extremely low in easily accessible tissue or body fluids. To circumvent these problems and develop an effective premortem diagnostic method, an alternative strategy is to identify proteins (other than PrPSc) that are indicative of disease and allow early detection of prion infection, also known as surrogate biomarkers, in easily accessible body fluids. This review focuses on recent efforts to identify potential diagnostic biomarkers by proteomic technologies, the current challenges and possible new directions for biomarker discovery and validation in the future.


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