cording to the manufacturer’s instructions. Optical density was measured at 450 nm in an ELISA micro-plate reader. Values were expressed as concentration per ml urine (albumin) or normalized to albumin or creatinine (a1-microglobulin). Urinary creatinine Measurement of urinary creatinine was performed using a commercial kit (Cayman Europe, Estonia) based on the Jaffe alkaline picrate reaction according to the manufacturer’s instruc- tions. Urine samples were diluted 1:15 in molecular biology grade water such that assay values were contained within the measurable range of the standard curve. Optical density was measured at 490 nm in an ELISA micro-plate reader. Values were expressed as concentration per ml urine. Western blots Total protein concentration was determined by bicinchoninic acid (BCA) or microBCA (Perbio Science, Switzerland). Equal amounts (mg) of total protein were separated by electrophoresis through 12% SDS-PAGE gels and transferred to nitrocellulose membranes. Reducing conditions were used in all cases except for detection of human a1-ACT. The membranes were blocked in PBST containing 5% non-fat milk for 2 h at room temperature prior to incubating overnight at 4uC with primary antibody. Following six minute washes with PBST the membranes were incubated in PBST containing 5% non-fat milk and the appropriate secondary antibody for 1 h at room temperature. In the case of cattle urine, Top-block (GE Healthcare, UK) was used instead of 5% non-fat milk. As a positive control, purified recombinant human a1-ACT (R&D Systems, UK) was also included. For detection of a1-ACT in human, sheep and deer urine, murine anti-human a1-ACT (R&D Systems, UK) was used at a concentration of 2 mg/ml followed by incubation with HRP- conjugated rabbit anti-mouse IgG2a (Invitrogen, UK) at a dilution of 1:5000. For detection of a1-ACT in cattle urine, rabbit anti- human a1-ACT (Dako, Switzerland) was used at a dilution of 1:1000, followed by incubation with HRP-conjugated donkey anti- rabbit IgG (GE Healthcare, UK) at a dilution of 1:1000. For detection of cystatin C in human CSF, samples were prepared under non-reducing conditions, electrophoresed and transferred to nitrocellulose membranes. After blocking as outlined above, membranes were incubated in PBST containing 5% non-fat milk and a 1:1000 dilution of rabbit anti-human cystatin C (Upstate, UK), followed by washing with PBST and incubation with HRP- conjugated donkey anti-rabbit IgG (GE Healthcare, UK) at a dilution of 1:1000. Following addition of ECL substrate (Perbio, Switzerland) signal was visualized using a VersaDoc model 4000 Imaging system (BioRad, Switzerland). Membranes were stripped (Western blotting stripping solution, Socochim, Switzerland) and re-probed with rabbit anti-human albumin (Abcam, UK), at a dilution of 1:5000, followed by HRP-conjugated donkey anti- rabbit IgG (GE Healthcare, UK), at a dilution of 1:1000, and subsequent ECL detection as outlined above. Body fluid samples of patients Samples were obtained from patients who presented to the NRPE, the United Kingdom CJD Surveillance Unit (CJDSU), the German Reference Centre for Prion Diseases, the Clinic for Nephrology, the Institute for Clinical Pathology, and the Division of Psychiatric Research at University of Z u¨rich. Collection of blood, CSF and urine for subsequent analysis was approved by the Swiss Federal Office of Health. Prion Disease Biomarker For control, we assembled two distinct groups of persons. Control group A consisted of individuals whose age and gender matched those of patients suffering from sCJD or AD, whereas the demographics of control group B matched patients suffering from variant CJD (vCJD). Specifically, control group B consisted of patients who initially presented to the UK CJDSU with suspected CJD, but were later determined to suffer from other ailments. Urine and CSF samples were obtained from patients at varying clinical stages of CJD, from mid-clinical stage to terminal disease. In the case of terminal patients, body fluids were collected at post- mortem. Plasma was derived from patients ante mortem. From pre- mortem patients where sCJD was suspected, samples were included in the study only following post-mortem confirmation of sCJD. In addition, we investigated a control group consisting of urine collected from patients post-kidney transplant following diabetic nephropathy, focal segmental glomerulosclerosis and reflux nephropathy conditions, from patients presenting with bladder carcinoma and bacteriuria, and from patients with cerebrovascu- lar disease or encephalitis. Urine from patients with known macro- or microhaematuria, or proteinuria, was excluded from further analysis. In retrospectively collected urine samples in which this was unknown, samples containing .150 mg/ml albumin were excluded from the study. Within approximately 18 h of collection, urine was centrifuged briefly, aliquoted and stored at 280uC until analysis. Blood was collected in tubes containing citrate to a final concentration of 0.5% and, within 12 h of collection, centrifuged a 2 280 g for 4 minutes at room temperature. The plasma layer was aliquoted and stored at 280uC until required. Cerebrospinal fluid was collected, aliquoted and stored at 280uC. Informed consent was obtained in all relevant cases. Field Animals Urine samples were obtained from sheep scrapie cases both pre- terminal (without the use of diuretic drugs or catheter) or at post- mortem via bladder puncture. Urine from deer CWD and cattle BSE cases (and controls) were obtained at post-mortem by bladder puncture. Pre-terminal scrapie cases consisted of six healthy Sarda sheep held in the Sassari region of Italy (PRNP genotypes: ARR/ ARQ, ARQ/VRQ, ARR/ARR, ARQ/ARQ) and one natural scrapie positive animal (ARQ/ARQ), from which urine was collected pre-clinical and throughout the clinical phase of disease. Urine from terminal sheep was derived from UK sheep of the Suffolk breed and consisted of males and females displaying no clinical signs of scrapie (ARR/ARR and ARQ/ARQ PRNP genotypes) and natural scrapie positive males and females (ARQ/ ARQ PRNP genotypes). Statistical tests Data was analyzed for statistical significance using two-tailed unpaired Mann-Whitney t-test with 95% confidence interval. Significance of p,0.05, and p,0.001 is indicated by * and ** respectively. Receiver operator characteristic (ROC) curves were calculated and plotted using the software MedCalc. Supporting Information Table S1 Found at: doi:10.1371/journal.pone.0003870.s001 (0.08 XLS) MB Acknowledgments We thank the Veterinary Laboratories Agency TSE archive for provision of urine from field cases of BSE-affected and unaffected cattle, Gabriella PLoS ONE | www.plosone.org 12 December 2008 | Volume 3 | Issue 12 | e3870