Prion Disease Biomarker Figure 4. Urinary a1-ACT of sCJD patients. (A) Equivalent amounts (50 mg) of urinary protein from sCJD patients and appropriate age-matched controls were fractionated under non-reducing SDS-PAGE conditions and subsequently analyzed by Western blot using an anti-human a1-ACT monoclonal antibody. A duplicate blot incubated with HRP-labeled secondary antibody alone is shown. To verify equivalent protein loading, blots were stripped and re-probed with anti-albumin antibodies. Molecular weight marker positions are indicated. a1-ACT levels are significantly higher in sCJD patient urine, corroborating the data obtained by ELISA measurement. (B) ELISA measurement of a1-ACT in urine collected sequentially during the clinical phase of disease of two sCJD patients (represented as circles or triangles) indicates a progressive increase in a1-ACT levels normalized both for albumin and creatinine. Error bars represent the standard deviations in replicate measurements of each sample. doi:10.1371/journal.pone.0003870.g004 observations suggest that a1-ACT elevation occurs in prion infections of many species. Serpin-a3n encodes a secretory leader peptide, and a1-ACT is present in body fluids. This raised the possibility that a1-ACT may represent a candidate biomarker of prion infection. Since many brain metabolites are found in the CSF, we first tested whether CSF concentrations of a1-ACT would predict prion infection. Indeed, sCJD patients exhibited a highly significant (p,0.001) increase of a1-ACT in CSF when normalized against total CSF protein and compared to appropriate age-matched controls. a1-ACT is elevated in brains of AD patients and contributes to AD amyloid plaque deposits [17,18], but the value of a1-ACT as a biomarker of AD is unclear. Some studies have reported elevated a1-ACT in plasma or CSF of AD patients [19,20,21,22], whereas others found no changes [29]. In agreement with the latter study, we found no increased a1-ACT in plasma and CSF of AD patients in the present study. We then sought to compare the diagnostic value of a1-ACT with that of cystatin C, which had been claimed to be a biomarker for CJD. We found that plasma and CSF levels of cystatin C did not discriminate between AD patients and age and gender-matched control patients, in agreement with previous findings [30]. Also, CSF measurements of cystatin C by sandwich ELISA in large collectives of sCJD, vCJD, and control samples was not discrim- inatory between these three groups. The latter finding is at striking variance with two studies of cystatin C in the CSF of small collectives of CJD patients (n = 8 and 6, respectively) [15,16] reporting up to 30-fold increases of CSF cystatin C in sCJD patients. Cystatin C undergoes proteolytic processing, and a 12.5 kDa cleavage product was reported to be elevated in the CSF of patients suffering from multiple sclerosis, even if the levels of full- length 13.4 kDa cystatin C were unaltered [31]. Therefore, antibodies differentially recognizing cystatin C and its degradation products may introduce a confounding factor. However, we were unable to demonstrate any differences in cystatin C levels in CSF of sCJD patients by Western blotting even when using the same antibody that had led to the claim of a 30-fold elevation of CSF cystatin C in sCJD patients [16]. We conclude that CSF cystatin C is unaltered in the collective of patients investigated here (n = 8) and therefore does not represent a biomarker of sCJD or vCJD. PLoS ONE | www.plosone.org 7 December 2008 | Volume 3 | Issue 12 | e3870