Prion Disease Biomarker Figure 5. Receiver Operating Characteristic (ROC) Curve Analysis. ROC curves were calculated for mg a1-ACT/mg albumin (a & c) or mg a1- ACT/mg creatinine (b & d) for sCJD patients relative to either Control group A and AD patients (a & b) or Control group A, AD and Control C patients (b & d). Sensitivity, specificity, and threshold (criterion) for each are indicated. doi:10.1371/journal.pone.0003870.g005 We then studied the occurrence of immunoglobulins, a1-ACT, and cystatin C in urine samples. We were unable to confirm the previously reported presence of protease-resistant immunoglobulin light chains in the urine of prion-affected animals and humans [32,33,34]. Our use of anti-mouse IgG2a as secondary antibody rather than total IgG may account for this discrepancy. Also, cystatin C excretion was unaltered urine of AD, sCJD, and vCJD patients regardless of the normalization method used, and therefore not diagnostic of prion infection. Surprisingly, urine samples from sCJD patients belonging to the UK, German and Swiss collectives displayed consistently elevated a1-ACT. Significance was attained independently of whether ELISA results were expressed as raw concentrations, or normal- ized against creatinine or albumin. ROC curve analysis confirms PLoS ONE | www.plosone.org 8 December 2008 | Volume 3 | Issue 12 | e3870