A Pitfall in Diagnosis of Human Prion Diseases Using Detection of Protease-resistant Prion Protein in Urine

Abstract

Because a definite diagnosis of prion diseases relies on the detection of the abnormal isoform of prion protein (PrP Sc ), it has been urgently necessary to establish a non-invasive diagnostic test to detect PrP Sc in human prion diseases. To evaluate diagnostic usefulness and reliability of the detection of protease-resistant prion protein in urine, we extensively analyzed proteinase K (PK)-resistant proteins in patients affected with prion diseases and control subjects by Western blot, a coupled liquid chromatography and mass spectrometry analysis, and N-terminal sequence analysis. The PK-resistant signal migrating around 32 kDa previously reported by Chem. 276, 31479 -31482) was not observed in this study. Instead, discrete protein bands with an apparent molecular mass of ϳ37 kDa were detected in the urine of many patients affected with prion diseases and two diseased controls. Although these proteins also gave strong signals in the Western blot using a variety of anti-PrP antibodies as a primary antibody, we found that the signals were still detectable by incubation of secondary antibodies alone, i.e. in the absence of the primary antiPrP antibodies. Mass spectrometry and N-terminal protein sequencing analysis revealed that the majority of the PK-resistant 37-kDa proteins in the urine of patients were outer membrane proteins (OMPs) of the Enterobacterial species. OMPs isolated from these bacteria were resistant to PK and the PK-resistant OMPs from the Enterobacterial species migrated around 37 kDa on SDS-PAGE. Furthermore, nonspecific binding of OMPs to antibodies could be mistaken for PrP Sc . These findings caution that bacterial contamination can affect the immunological detection of prion protein. Therefore, the presence of Enterobacterial species should be excluded in the immunological tests for PrP Sc in clinical samples, in particular, urine.

Characterization of the Prion Protein in Human Urine

“…They also described the pres-ence of protease-sensitive PrP, apparently full-length PrP C , in controls that were free of prion disease, which suggests that normal urine contains PrP C whereas urine from individuals affected with prion disease also contains PrP Sc . However, three subsequent studies using the same antibody failed to detect PrP in urine from normal and prion disease-affected individuals and demonstrated that the false positive results arose from the cross-reaction of anti-mouse IgG with either contaminating bacterial proteins (27) or urinary IgG fragments (28,29). Nonetheless, a series of more recent studies have observed prion infectivity in urine from experimentally and naturally affected animals (30 -33).…”

Evaluation of Confocal Fluorescence Spectroscopy for the Detection of Pathological Prion Proteins

“…However, a routine pre-symptomatic test for TSEs would require examination of easily accessible body fluids such as urine, saliva or blood. Although, the presence of PrP Sc -like molecules has been reported in urine of TSE infected animals and humans [24], this report has been questioned by results showing cross-reactivity of anti-PrP antibodies with bacterial proteins present in urine [25]. Because prions were found in the blood of scrapie infected sheep [12], blood seems the best option as a matrix for a non-invasive TSE test for live animals and humans.…”

Current and Developing Technologies for Monitoring Agents of Bioterrorism and Biowarfare

“…For example, Western blots of urine samples used to detect the presence of protease-resistant prion proteins could be misinterpreted. Bands with a molecular mass similar to prion proteins can be detected from these Western blots (96).…”