Automated Competitive Protein-Binding Assay for Total 25-OH Vitamin D, Multicenter Evaluation and Practical Performance.

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 DEQAS 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.

Novel multiplex technology for diagnostic characterization of rheumatoid arthritis.

INTRODUCTION: The aim of this study was to develop a clinical-grade, automated, multiplex system for the differential diagnosis and molecular stratification of rheumatoid arthritis (RA). METHODS: We 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. RESULTS: We 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%. CONCLUSIONS: The 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.

Novel enzymatic activity derived from the Semliki Forest virus capsid protein.

The N-terminal segment of the Semliki Forest virus polyprotein is an intramolecular serine protease that cleaves itself off after the invariant Trp267 from a viral polyprotein and generates the mature capsid protein. After this autoproteolytic cleavage, the free carboxylic group of Trp267 interacts with the catalytic triad (His145, Asp167 and Ser219) and inactivates the enzyme. We have deleted the last 1-7 C-terminal residues of the mature capsid protease to investigate whether removal of Trp267 regenerates enzymatic activity. Although the C-terminally truncated polypeptides do not adopt a defined three-dimensional structure and show biophysical properties observed in natively unfolded proteins, they efficiently catalyse the hydrolysis of aromatic amino acid esters, with higher catalytic efficiency for tryptophan compared to tyrosine esters and k(cat)/K(M) values up to 5 x 10(5) s(-1) M(-1). The enzymatic mechanism of these deletion variants is typical of serine proteases. The pH enzyme activity profile shows a pK(a1)=6.9, and the Ser219Ala substitution destroys the enzymatic activity. In addition, the fast release of the first product of the enzymatic reaction is followed by a steady-state second phase, indicative of formation and breakdown of a covalent acyl-enzyme intermediate. The rates of acylation and deacylation are k(2)=4.4+/-0.6 s(-1) and k(3)=1.6+/-0.5 s(-1), respectively, for a tyrosine derivative ester substrate, and the amplitude of the burst phase indicates that 95% of the enzyme molecules are active. In summary, our data provide further evidence for the potential catalytic activity of natively unfolded proteins, and provide the basis for engineering of alphavirus capsid proteins towards hydrolytic enzymes with novel specificities.

Characterization of recombinant, membrane-attached full-length prion protein.

An abnormal isoform, PrP(Sc), 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 C-terminal 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.

Folding and intrinsic stability of deletion variants of PrP(121-231), the folded C-terminal domain of the prion protein.

Transmissible spongiform encephalopathies in mammals are believed to be caused by PrPSc, the insoluble, oligomeric isoform of the cellular prion protein PrPC. PrPC and the subunits of PrPSc have identical covalent but different tertiary structure. To address the question of whether parts of the structure of PrPC are sufficiently stable to be retained in PrPSc, we have constructed two deletion variants of the C-terminal PrPC domain, PrP(121-231), which is the only part of recombinant PrP with defined tertiary structure. One of the variants, H2-H3, comprises the last two alpha-helices of PrP(121-231) that have been proposed to be preserved in models of PrP(Sc). In the other variant, PrP(121-231)-deltaH1, the first alpha-helix of PrP(121-231) was deleted and replaced by introduction of the beta-turn dipeptide Asn-Gly between the strands of the single beta-sheet of PrP(121-231). Although both deletion constructs still show alpha-helical CD-spectra, they are more disordered and thermodynamically strongly destabilized compared to PrP(121-231), with free energies of folding close to zero. These data demonstrate that the tertiary structure context is critical for the conformation of the segment comprising alpha-helix 2 and 3 in the solution structure of recombinant PrP.