TY - GEN A1 - Hodoroaba, Vasile-Dan A1 - Hörenz, Christoph T1 - Improving nanoparticle size measurement accuracy for safety assessment T2 - EURAMET News N2 - Nanomaterials and nanoparticles are finding applications across a wide range of technology sectors, from medicine and food to transportation and construction. In order to assess these new materials for potential risks to health and the environment, they need to be well-characterised. The measurement of constituent nanoparticle size, shape, and size distribution are important factors for the risk evaluation process. EMPIR project Improved traceability chain of nanoparticle size measurements (17NRM04, nPSize) is working to assess a range of traceable nanoparticle measurement approaches, including Scanning Electron Microscopy (also in Transmission Mode), Atomic Force Microscopy and Small Angle X-ray Scattering, and deliver improved calibration methods to users. For the techniques under investigation, physical models of their response to a range of nanoparticle types are developed. Validated reference materials will also be used for an inter-comparison of measurement systems, with an evaluation of the associated measurement uncertainty. With project contributions to standards development work, manufacturers will be better placed to assess the human and environmental risks posed by nanomaterials across a whole range of products. KW - Nanoparticles KW - Particle size distribution KW - Traceability KW - Electron microscopy KW - AFM KW - SAXS PY - 2021 UR - https://www.euramet.org/?L=0&news=40%3A1159 SP - 1 PB - EURAMET e.V. CY - Braunschweig AN - OPUS4-52129 LA - eng AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER - TY - GEN A1 - Tchipilov, Teodor A1 - Meyer, Klas A1 - Weller, Michael G. T1 - Quantitative 1H Nuclear Magnetic Resonance (qNMR) of Aromatic Amino Acids for Protein Quantification T2 - Preprints N2 - qNMR is a valuable technique for metrological studies due to the uniformity of its signal response for all chemical species of an isotope of interest, which enables compound-independent calibration. However, protein quantification remained challenging as large molecules produce wide, low-intensity signals that reduce the already low sensitivity. Combining qNMR with the hydrolysis of protein samples into amino acids circumvents many of these issues and facilitates the use of NMR spectroscopy for absolute protein and peptide quantification.In this work, different conditions have been tested for quantifying aromatic amino acids and proteins. First, we examined the pH-based signal shifts in the aromatic region. The preferable pH depends on the selection of the amino acids for quantification and which internal standard substance should be used to avoid peak overlap. Several aromatic compounds, such as terephthalic acid, sulfoisophthalic acid, and benzene tricarboxylic acid, have been applied as internal standards. The quantification of amino acids from an amino acid standard, as well as from a certified reference material (bovine serum albumin), was performed. Using the first two suggested internal standards, recovery was ~ 97 % for histidine, phenylalanine, and tyrosine at a concentration of approximately 1 mM in solution. Acidic hydrolysis of a certified reference material (CRM) of bovine serum albumin (BSA) and subsequent quantification of Phe and Tyr yielded recoveries of 98 ± 2 and 88 ± 4 %, respectively, at a protein concentration of 16 g/L or 250 µM. KW - Amino acid analysis KW - AAA KW - Protein hydrolysis KW - Metrology KW - Traceability KW - Reference materials KW - Internal standards KW - Calibration PY - 2022 UR - https://nbn-resolving.org/urn:nbn:de:kobv:b43-564520 DO - https://doi.org/10.20944/preprints202211.0569.v1 SP - 1 EP - 13 PB - MDPI CY - Basel AN - OPUS4-56452 LA - eng AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER - TY - GEN A1 - Tscheuschner, Georg A1 - Kaiser, Melanie N. A1 - Lisec, Jan A1 - Beslic, D. A1 - Muth, Thilo A1 - Krüger, M. A1 - Mages, H.-W. A1 - Dorner, B. G. A1 - Knospe, J. A1 - Schenk, J. A. A1 - Sellrie, F. A1 - Weller, Michael G. T1 - MALDI-TOF-MS-based identification of monoclonal murine anti-SARS-CoV-2 antibodies within one hour T2 - Preprints N2 - During the SARS-CoV-2 pandemic, many virus-binding monoclonal antibodies have been developed for clinical and diagnostic purposes. This underlines the importance of antibodies as universal bioanalytical reagents. However, little attention is given to the reproducibility crisis that scientific studies are still facing to date. In a recent study, not even half of all research antibodies mentioned in publications could be identified at all. This should spark more efforts in the search for practical solutions for the traceability of antibodies. For this purpose, we used thirty-five monoclonal antibodies against SARS-CoV-2 to demonstrate how sequence-independent antibody identification can be achieved by simple means applied onto the protein. First, we examined the intact and light chain masses of the antibodies relative to the reference material NIST-mAb 8671. Already half of the antibodies could be identified based solely on these two parameters. In addition, we developed two complementary peptide mass fingerprinting methods with MALDI-TOF-MS that can be performed in 45 minutes and had a combined sequence coverage of over 80%. One method is based on the partial acidic hydrolysis of the protein by 5 mM of sulfuric acid at 99 °C. Furthermore, we established a fast way for a tryptic digest without an alkylation step. We were able to show that the distinction of clones is possible simply by a brief visual comparison of the mass spectra. In this work, two clones originating from the same immunization gave the same fingerprints. Later, a hybridoma sequencing confirmed the sequence identity of these sister clones. In order to automate the spectral comparison for larger libraries of antibodies, we developed the online software ABID 2.0 (https://gets.shinyapps.io/ABID/). This open-source software determines the number of matching peptides in the fingerprint spectra. We propose that publications and other documents critically relying on monoclonal antibodies with unknown amino acid sequences should include at least one antibody fingerprint. By fingerprinting an antibody in question, its identity can be confirmed by comparison with a library spectrum at any time and context. KW - SARS-CoV-2 antibody KW - Reproducibility crisis KW - Peptide mass fingerprinting KW - Monoclonal antibody KW - Traceability KW - Antibody identification KW - Identity KW - Antibody light chain KW - MALDI-TOF-MS KW - Trypsin KW - Acidic cleavage KW - Antibody subclass KW - Database KW - Peak overlap KW - ABID KW - Sulfuric acid KW - Online software KW - Sequencing KW - Peptide coverage PY - 2022 UR - https://nbn-resolving.org/urn:nbn:de:kobv:b43-545182 DO - https://doi.org/10.20944/preprints202203.0229.v1 SN - 2310-287X SP - 1 EP - 24 PB - MDPI CY - Basel AN - OPUS4-54518 LA - eng AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER - TY - GEN A1 - Tscheuschner, Georg A1 - Schwaar, Timm A1 - Weller, Michael G. T1 - Fast Confirmation of Antibody Identity by MALDI-TOF-MS Fingerprints T2 - Preprints N2 - Thousands of antibodies for diagnostic and other analytical purposes are on the market. However, it is often difficult to identify duplicates, reagent changes, and to assign the correct original publications to an antibody. This slows down scientific progress and might even be a cause of irreproducible research and a waste of resources. Recently, activities were started to suggest the sole use of recombinant antibodies in combination with the open communication of their sequence. In this case, such uncertainties should be eliminated. Unfortunately, this approach seems to be rather a long-term vision since the development and manufacturing of recombinant antibodies remain quite expensive in the foreseeable future. Also, nearly all commercial antibody suppliers may be reluctant to publish the sequence of their antibodies, since they fear counterfeiting. De-novo sequencing of antibodies is also not feasible today for a reagent user without access to the hybridoma clone. Nevertheless, it seems to be crucial for any scientist to have the opportunity to identify an antibody undoubtedly to guarantee the traceability of any research activity using antibodies from a third party as a tool. For this purpose, we developed a method for the identification of antibodies based on a MALDI-TOF-MS fingerprint. To circumvent lengthy denaturation, reduction, alkylation, and enzymatic digestion steps, the fragmentation was performed with a simple formic acid hydrolysis step. Eighty-nine unknown monoclonal antibodies were used for this study to examine the feasibility of this approach. Although the molecular assignment of peaks was rarely possible, antibodies could be easily recognized in a blinded test, simply from their mass-spectral fingerprint. A general protocol is given, which could be used without any optimization to generate fingerprints for a database. We want to propose that in most scientific projects relying critically on antibody reagents, such a fingerprint should be established to prove and document the identity of the used antibodies and to assign a specific reagent to a datasheet of a commercial supplier, a public database record or an antibody ID. KW - Reproducibility KW - Quality Control KW - Traceability KW - Diagnostics KW - ELISA KW - Immunoassay PY - 2020 UR - https://nbn-resolving.org/urn:nbn:de:kobv:b43-506590 DO - https://doi.org/10.20944/preprints202002.0207.v1 SN - 2310-287X SP - 1 EP - 17 PB - MDPI CY - Basel AN - OPUS4-50659 LA - eng AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER - TY - GEN A1 - Vogl, Jochen T1 - Roadmap for the purity determination of pure metallic elements – Basic rinciples and helpful advice T2 - BIPM Webpage N2 - High purity materials can serve as a realisation of the Système International d’Unitès (SI) unit amount of substance for the specific element. Solutions prepared from such high purity materials using gravimetric preparation and the concept of molar mass are used as calibration solutions in many fields of analytical chemistry. Calibration solutions prepared this way provide the traceability to the SI and are the metrological basis in elemental analysis. The preparation and characterization of such primary pure substances, representing the realisation of the SI unit amount of substance, is undertaken only by a small number of National Metrology Institutes (NMI) and Designated Institutes (DI). Many other NMIs and DIs, however, prepare elemental calibration solutions as calibrants for their measurement services, such as the certification of matrix Reference Materials or the provision of reference values for Proficiency Testing schemes. The elemental calibration solutions used for this purpose are not a direct service to customers, such as preparing secondary calibration solutions, but provide the source of traceability for the other services. Hence, it is necessary for the NMI or DI to obtain data on the purity of the pure metals or other materials used to prepare the solutions with measurement uncertainties meeting the needs of the above described services. This is commonly undertaken as a “fit for purpose” assessment, appropriate for the uncertainty requirement of the service provided to customers. As a consequence, total purity measurements are a long-term strategy of CCQM-IAWG. Several studies were conducted (CCQM-P107, CCQM-K72 and CCQM-P149) on the measurement of the purity of zinc. From these studies, several conclusions can be drawn for the purity assessment of a pure (metallic) element. These conclusions will be put together in this document in order to assist all NMIs/DIs in performing a purity assessment, whenever needed. KW - Purity assessment KW - Metal assay approach KW - Impurity assessment approach KW - Traceability PY - 2018 UR - https://www.bipm.org/wg/CCQM/IAWG/Allowed/April_2017/CCQM-IAWG17-28.pdf.pdf SP - 1 EP - 9 CY - Sevre AN - OPUS4-46834 LA - eng AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER -