IntroductionThe aim of this study was to develop a clinical-grade, automated, multiplex system for the differential diagnosis and molecular stratification of rheumatoid arthritis (RA).MethodsWe profiled autoantibodies, cytokines, and bone-turnover products in sera from 120 patients with a diagnosis of RA of < 6 months' duration, as well as in sera from 27 patients with ankylosing spondylitis, 28 patients with psoriatic arthritis, and 25 healthy individuals. We used a commercial bead assay to measure cytokine levels and developed an array assay based on novel multiplex technology (Immunological Multi-Parameter Chip Technology) to evaluate autoantibody reactivities and bone-turnover markers. Data were analyzed by Significance Analysis of Microarrays and hierarchical clustering software.ResultsWe developed a highly reproducible, automated, multiplex biomarker assay that can reliably distinguish between RA patients and healthy individuals or patients with other inflammatory arthritides. Identification of distinct biomarker signatures enabled molecular stratification of early-stage RA into clinically relevant subtypes. In this initial study, multiplex measurement of a subset of the differentiating biomarkers provided high sensitivity and specificity in the diagnostic discrimination of RA: Use of 3 biomarkers yielded a sensitivity of 84.2% and a specificity of 93.8%, and use of 4 biomarkers a sensitivity of 59.2% and a specificity of 96.3%.ConclusionsThe multiplex biomarker assay described herein has the potential to diagnose RA with greater sensitivity and specificity than do current clinical tests. Its ability to stratify RA patients in an automated and reproducible manner paves the way for the development of assays that can guide RA therapy.

An abnormal isoform, PrPSc , of the normal cellular prion protein (PrP C ) is the major component of the causative agent of prion diseases. Both isoforms were found to possess the same covalent structures, including a Cterminal glycosylphosphatidylinositol anchor, but different secondary and tertiary structures. In this study, a variant of full-length PrP with an unpaired cysteine at the C terminus was recombinantly produced in Escherichia coli, covalently coupled to a thiol-reactive phospholipid, and incorporated into liposomes to serve as a model for studying possible changes in structure and stability of recombinant PrP upon membrane attachment. Covalent coupling of PrP to liposomes did not result in significant structural changes observable by far-UV circular dichroism. Moreover, limited proteolysis experiments failed to detect changes in the stability of liposome-bound PrP relative to soluble PrP. These data suggest that the requirement of raft localization for the PrP C to PrP Sc conversion, observed previously in cell culture models, is not because of a direct influence of raft lipids on the structure and stability of membranebound PrP C but caused by other factors, e.g. increased local PrP concentrations or high effective concentrations of membrane-associated conversion factors. The availability of recombinant PrP covalently attached to liposomes provides the basis for systematic in vitro conversion assays with recombinant PrP on the surface of membranes. In addition, our results indicate that the three-dimensional structure of mammalian PrP C in membranes is identical to that of recombinant PrP in solution.

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Background:The Roche Elecsys Vitamin D Total competitive protein-binding assay uses recombinant vitamin D binding protein for measuring 25-hydroxyvitamin D (25-OHD), which is different from commonly used antibody assays. Methods: The assay, standardized against LC-MS/MS, was tested at four sites. Evaluation included precision; between-laboratory variability; functional sensitivity; correlation to LC-MS/MS, HPLC, and immunoassays; as well as robustness, traceability, and EQAS performance. Results: Precision testing showed within-run coefficient of variations (CVs) of ࣘ7%, within-laboratory CVs of <9.5%, between-laboratory precision CVs of ࣘ10.1%, and a functional sensitivity below 9.8 nmol/l (at CV 12.9%). The assay showed equivalent 25-OHD levels for matched serum and plasma samples, good reagent lot-to-lot consistency in pooled sera over time, and good agreement with HPLC (relative bias −8.8%). Comparison with LC-MS/MS methods yielded relative biases of −15.4, −13.5, −10.2, and 3.2%. Comparison against immunoassays showed a relative bias of 14.5% (DiaSorin Liaison) and −58.2% (IDS-iSYS). The overall mean results in 2 years DE-QAS was 102% of the ALTM. In a certified reference patient panel, the average bias was <4% for the sum of 25-OHD2 and 25-OHD3. Conclusion: The Elecsys Vitamin D Total assay demonstrated good overall performance and is, according to present standards, very suitable for automated measurement of 25-OHD.