First amyloid ß1-42 certified reference material for re-calibrating commercial immunoassays.

INTRODUCTION: Reference materials based on human cerebrospinal fluid were certified for the mass concentration of amyloid beta (Aß)1-42 (Aß42 ). They are intended to be used to calibrate diagnostic assays for Aß42 . METHODS: The three certified reference materials (CRMs), ERM-DA480/IFCC, ERM-DA481/IFCC and ERM-DA482/IFCC, were prepared at three concentration levels and characterized using isotope dilution mass spectrometry methods. Roche, EUROIMMUN, and Fujirebio used the three CRMs to re-calibrate their immunoassays. RESULTS: The certified Aß42 mass concentrations in ERM-DA480/IFCC, ERM-DA481/IFCC, and ERM-DA482/IFCC are 0.45, 0.72, and 1.22 µg/L, respectively, with expanded uncertainties (k = 2) of 0.07, 0.11, and 0.18 µg/L, respectively. Before re-calibration, a good correlation (Pearson's r > 0.97), yet large biases, were observed between results from different commercial assays. After re-calibration the between-assay bias was reduced to < 5%. DISCUSSION: The Aß42 CRMs can ensure the equivalence of results between methods and across platforms for the measurement of Aß42 .

Gaps in the traceability chain of human growth hormone measurements.

BACKGROUND: Human growth hormone (hGH) is measured for the diagnosis of secretion disorders. These measurements fall under the EU Directive 98/79/EC on in vitro diagnostic medical devices requiring traceability of commercial calibrator values to higher-order reference materials or procedures (Off J Eur Communities 1998 Dec 7;L 331:1-37). External quality assessment schemes show large discrepancies between results from different methods, even though most methods provide results traceable to the recommended International Standard (IS 98/574). The aim of this study was to investigate possible causes for these discrepancies. METHODS: We investigated the commutability and recovery of hGH in reconstituted IS 98/574. We tested different reconstitution protocols and used 4 different serum matrices for spiking. These IS preparations were measured together with serum samples. We quantified hGH by 5 different methods in 4 different laboratories. RESULTS: Results from the different methods correlated well for the serum samples. Mean discrepancies between results from different methods were ≤20%. None of the IS preparations was commutable for all the method comparisons. The recovery of hGH in preparations of IS 98/574 depended on the reconstitution protocol (>10-fold differences) and background matrix (relative differences ≤17% for different serum matrices). CONCLUSIONS: The use of different protocols for reconstitution and spiking of hGH reference preparations affects quantification by immunoassays, potentially leading to a bias between commercial methods, despite the use of calibrators with values claimed to be traceable to the same higher-order reference material.

Structure and nuclear import function of the C-terminal domain of influenza virus polymerase PB2 subunit.

The trimeric influenza virus polymerase, comprising subunits PA, PB1 and PB2, is responsible for transcription and replication of the segmented viral RNA genome. Using a novel library-based screening technique called expression of soluble proteins by random incremental truncation (ESPRIT), we identified an independently folded C-terminal domain from PB2 and determined its solution structure by NMR. Using green fluorescent protein fusions, we show that both the domain and the full-length PB2 subunit are efficiently imported into the nucleus dependent on a previously overlooked bipartite nuclear localization sequence (NLS). The crystal structure of the domain complexed with human importin alpha5 shows how the last 20 residues unfold to permit binding to the import factor. The domain contains three surface residues implicated in adaptation from avian to mammalian hosts. One of these tethers the NLS-containing peptide to the core of the domain in the unbound state.

Nuclear traffic of influenza virus proteins and ribonucleoprotein complexes.

Influenza virus is a negative strand RNA virus and is one of the rare RNA viruses to replicate in the nucleus. The viral RNA is associated with 4 viral proteins to form ribonucleoprotein particles (RNPs). After cell entry the RNPs are dissociated from the viral matrix protein in the low pH of the endosome and are actively imported into the cell nucleus. After translation of viral mRNAs, the proteins necessary for the assembly of new RNPs (the nucleoprotein and the three subunits of the polymerase complex) are also imported into the nucleus. Apart from these four proteins, part of the newly made matrix protein is also imported and the nuclear export protein (NEP) enters the nucleus probably through diffusion. Finally, NS1 also enters the nucleus in order to regulate a number of nuclear processes. The nuclear localization signals on all these viral proteins and their interaction with the cellular transport system are discussed. In the nucleus, the matrix protein binds to the newly assembled RNPs and NEP then binds to the matrix protein. NEP contains the nuclear export signal necessary for transport of the RNPs to the cytoplasm, necessary for the budding of new virus particles. There appears to be a intricate ballet in exposing and hiding nuclear transport signals which leads to a unidirectional transport of the RNPs to the nucleus at the start of the infection process and an opposite unidirectional export of RNPs at the end of the infection.

Human importin alpha and RNA do not compete for binding to influenza A virus nucleoprotein.

Influenza virus has a segmented genome composed of eight negative stranded RNA segments. Each segment is covered with NP forming ribonucleoproteins (vRNPs) and carries a copy of the heterotrimeric polymerase complex. As a rare phenomenon among the RNA viruses, the viral replication occurs in the nucleus and therefore implies interactions between host and viral factors, such as between importin alpha and nucleoprotein. In the present study we report that through binding with the human nuclear receptor importin α5 (Impα5), the viral NP is no longer oligomeric but maintained as a monomer inside the complex. In this regard, Impα5 acts as a chaperone until NP is delivered in the nucleus for viral RNA encapsidation. Moreover, we show that the association of NP with the host transporter does not impair the binding of NP to RNA. The complex human Impα5-NP binds RNA with the same affinity as wt NP alone, whereas engineered monomeric NP through point mutations binds RNA with a strongly reduced affinity.