TY - GEN A1 - Hiller, Lukas T1 - Herstellung und Charakterisierung eines rekombinanten Anti-Tetanus-Antikörpers aus verschiedenen Säugerzelllinien N2 - Antikörper sind in der medizinischen Welt nicht mehr wegzudenken. Ihr Einsatz-Repertoire als Therapeutika für die Behandlung diverser Krankheiten und Infektionen weitet sich von Jahr zu Jahr mehr aus. Dabei basiert ein Großteil der verwendeten Antikörper zu diesem Zeitpunkt auf monoklonalen Antikörpern auf Basis von Hybridomazellen. Doch hier könnten rekombinante Antikörper aufgrund ihrer Vektor-basierten Expression eine vielversprechende und womöglich sogar revolutionierende Alternative bieten. So ergeben sich neue Optimierungsoptionen für Merkmale wie Affinität, Stabilität und Kompatibilität über Anpassungen auf genetischer oder posttranslationaler Ebene. Allerdings ergibt sich kein Anwendungszweck ohne die vorhergehende detaillierte Charakterisierung der Antikörper. Denn nur mit spezifischem Wissen über die diversen Eigenschaften und Wechselwirkungen kann eine effektive und präzise Verwendung in der Diagnostik und Therapeutik gewährleistet werden und auf diese Weise Leben retten. Aus diesem Grund beschäftigt sich diese Masterarbeit mit der Vertiefung der rekombinanten Antikörper-Thematik. Dazu soll ein Anti-Tetanus-Antikörper erstmals rekombinant in verschiedenen Zelllinien hergestellt und anschließend über umfangreiche analytische Verfahren mit einem Fokus auf die Einflussanalyse der unterschiedlichen posttranslationalen Glykosylierungen charakterisiert werden. Zu diesem Zweck wurden als Grundlage einerseits zwei P1316-Dual-Expressions-Vektoren mit variierenden Insert-Positionen der schweren und leichten Kette durch Gibson-Assembly sowie individuellen pRK5-Plasmide für eine separiert Expression der Antikkörperketten mithilfe Restriktionsfragment-Ligation generiert. In einer Gegenüberstellung der der Expressionsausbeuten stellte sich die Überlegenheit der P1316-Plasmid-Konfiguration heraus, in der das Leichtketten-Insert an der ersten Expressionsstelle positioniert ist. Deshalb wurde dieser Vektor als Ausgangspunkt für die anschließende hochskalierten Herstellung des Anti-Tetanus-Antikörpers in Expi293F-GnTI- und ExpiCHO-S-Zellen verwendet. Ein ELISA bestätigte die Bindungsaktivität der rekombinanten Antikörper am Tetanus-Toxoid unabhängig vom Expressionssystem. Nach einer folgenden Optimierung der für die Aufreinigung verwendeten Protein-A-Affinitätschromatographie, wurde über weitere Charakterisierungsmethoden wie Größenausschlusschromatographie und dynamische Lichtstreuung eine vergleichbare Aggregation aufgrund der sauren Elution aufgezeigt. In diesem Kontext wurde die Notwendigkeit einer Zweistufen-Präparation mit Affinitäts- und Größenausschlusschromatografie für den Erhalt monodisperser Proben verdeutlicht. Zudem wurden die verschiedenen intakten Massen mitsamt ihrer spezifischen Glykan-abhängigen Streumaßen in Relation zum monoklonalen NISTmAB-Referenzantikörper bestimmt. Dabei konnte den Erwartungen nach sowohl die geringere Masse als auch die uniformere Glykosylierung der in Expi293 GnTI- exprimierten Immunoglobuline in Relation zum ExpiCHO-S-Antikörper nachgewiesen werden. Anschließend wurde das Stabilitätsverhalten mittels thermischer Entfaltung und dynamischer Lichtstreuung über einen ausgeweiteten Zeitraum sowie verschiedenen Temperaturen dokumentiert. Grundlegend wurde eine höhere thermische Stabilität der zweiten konstanten Schwerketten-Domäne des aus ExpiCHO-S-Zellen stammenden Antikörpers nachgewiesen. Außerdem konnte eine problemlose Lagerungsfähigkeit beider unterschiedlich glykosylierter Antikörper bei Raumtemperatur für mindestens 4 Wochen festgestellt werden. Des Weiteren wurde die Langzeitlagerung im Rahmen der Auswirkung von Gefrier-Auftau-Zyklen mit gleichbleibenden Methoden sowie zusätzlicher Größenausschlusschromatographie erprobt. Hierbei stellte sich der Erhalt der strukturellen Integrität nach bis zu 15 Zyklen heraus. Allerdings geht die Monodispersität bereits nach wenigen Zyklen verloren, wobei die geringfügiger glykosylierten Expi293F-GnTI-Anti-Tetanus-Antikörper eine stärkere Beeinträchtigung durch multiple Gefrier-Auftau-Zyklen aufwiesen. KW - Rekombinante Antikörper KW - Affinitätschromatographie KW - Größenausschlusschromatographie KW - MALDI-TOF-MS KW - Dynamische Lichtstreuung PY - 2024 SP - 1 EP - 120 CY - Berlin AN - OPUS4-59865 LA - deu AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER - TY - GEN A1 - Gornushkin, Igor A1 - Kornev, R.A. A1 - Shabarova, L.V. A1 - Ermakov, A.A. A1 - Mochalov, G.M. A1 - Rekunov, N.V. A1 - Medov, V.A. A1 - Chaschina, A.D. A1 - Kalinina, A.A. T1 - Features of Plasma-Chemical Hydrogen Reduction of Volatile Silicon and Germanium Chlorides in RF-arc Discharge T2 - Preprints.org N2 - The processes of hydrogen reduction of silicon and germanium chlorides under the conditions of radio-frequency (40.68 MHz) counteracted arc discharge stabilized between two rod electrodes were investigated. The main gas-phase and solid products of plasma-chemical transformations were determined. Thermodynamic analysis of SiCl4 + H2 and GeCl4 + H2 systems was carried out. It is shown that under the implemented experimental conditions, equilibrium components of the products are established. The detected spectra of chemical activity were studied, which gave reason to assume that the molecular mechanism of the hydrogen reduction process is the main one. The impurity composition of gas-phase and solid reaction products was investigated. The possibility of single-stage production of high-purity Si and Ge mainly in the form of compact ingots, as well as high-purity chlorosilanes and trichlorogermane, was shown. KW - RF-arc discharge KW - Hydrogen reduction KW - Silicon chloride KW - Germanium chloride KW - Thermodynamic KW - Silicon KW - Germanium PY - 2023 UR - https://nbn-resolving.org/urn:nbn:de:kobv:b43-588909 DO - https://doi.org/10.20944/preprints202311.1412.v1 SP - 1 EP - 14 AN - OPUS4-58890 LA - eng AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER - TY - GEN A1 - Scholtz, Lena A1 - Resch-Genger, Ute T1 - Behind the Paper - Dual color pH probes made from silica and polystyrene nanoparticles and their performance in cell studies T2 - Springer Nature research communities - Community blogs for our authors, editors and all of the research community N2 - In this contribution we highlight the importance of comparison for scientific research while developing a new, functional pH sensor system, and the valuable insights this can provide. KW - Dye KW - Optical Spectroscopy KW - pH probe KW - Silica and Polystyrene Particles KW - Nano KW - Surface groups KW - Safe-by-Design KW - Cell studies KW - Sensors KW - Particle Synthesis KW - Fluorescence PY - 2023 UR - https://communities.springernature.com/posts/dual-color-ph-probes-made-from-silica-and-polystyrene-nanoparticles-and-their-performance-in-cell-studies SP - 1 EP - 2 PB - Springer Nature CY - London AN - OPUS4-59150 LA - eng AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER - TY - GEN A1 - Vergin, Chantal Celine T1 - Entwicklung eines Extraktionsverfahrens für Mykotoxine aus Lebensmitteln für die Messung in immunanalytischen Assays und Sensoren N2 - Mykotoxine sind sekundäre Stoffwechselprodukte, die von Schimmelpilzen gebildet werden. Sie sind unvermeidliche Lebensmittelkontaminanten und stellen daher ein ernstes Gesundheitsrisiko für Mensch und Tier dar. Aus diesem Grund hat die Europäische Kommission Vorschriften erlassen, die Höchstwerte für Mykotoxine in bestimmten Lebensmitteln vorsehen. Um die gesetzlichen Grenzwerte einzuhalten, müssen in der Lebensmittelindustrie Mykotoxinanalysen der eingehenden Waren durchgeführt werden. Gegenwärtig umfasst die Analyse zeitaufwändige Probenvorbereitungsschritte, denen in der Regel eine chromatographische Analyse folgt. Immunoassays ermöglichen eine Vor-Ort-Analyse, aber in der Regel sind für feste Lebensmittelproben lösungsmittelbasierte Extraktionsschritte erforderlich. Da Immunoassays in der Regel nicht mit den hohen Lösungsmittelkonzentrationen kompatibel sind, die üblicherweise für die Mykotoxinextraktion verwendet werden (z.B. Acetonitril oder Methanol), war es das Ziel dieser Arbeit, ein Extraktionsverfahren für die Mykotoxine Deoxynivalenol und Ochratoxin A aus Weizenmehl und Getreide zu entwickeln, das für Immunoassays und -sensoren verwendet werden kann. Vier verschiedene Lösungsmittel, Methanol (10 %), Ethanol (5 %) und Acetonitril (10 %) sowie Milli-Q-Wasser wurden für die Extraktion des Analyten Deoxynivalenol getestet. Um negative Umweltaspekte zu vermeiden und aufgrund der vergleichbaren Extraktionseffizienz von Milli-Q-Wasser gegenüber den anderen Lösungsmitteln, wurde Milli-Q-Wasser als Extraktionslösung im optimierten Extraktionsprotokoll für Deoxynivalenol verwendet. Es wurden verschiedene Extraktionsbedingungen, wie die Extraktionszeit, das Lösungsmittel-Feststoff-Verhältnis, der Filtrationsschritt, der Zentrifugationsschritt sowie die Zusammensetzung des Wassers untersucht. Auf der Grundlage der Ergebnisse wurde ein Extraktionsprotokoll festgelegt, mit dem Wiederfindungsraten in einem Bereich von x bis x erzielt wurden. Das Extraktionsprotokoll umfasste die Extraktion von 2 g Weizenmehl mit 40 ml Milli-Q-Wasser für 30 Minuten, die anschließende Zentrifugation bei 3000 U/min für 15 Minuten bei 4 °C und die Filtration des Überstands. KW - Mykotoxine KW - ELISA KW - Probenvorbereitung PY - 2023 SP - 1 EP - 81 CY - Berlin AN - OPUS4-59199 LA - deu 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 - Hartfiel, Reni T1 - Zweistufiges Screening von One-Bead-One-Peptide-Bibliotheken linearer & cyclischer Peptide gegen Pflanzenviren T2 - Masterarbeit N2 - In der Masterarbeit wurde ein Screening gegen den CCMV mit einer linearen Peptidbibliothek entwickelt, wobei zwei lineare Binder identifiziert wurden. Das resynthetisierte Peptid wurde auf seine Bindungseigenschaften mittels ELISA und MST untersucht. Aufgrund eines Aminosäurefehlers in der selbst durchgeführten Resynthese des Peptids sind die Ergebnisse nicht vollständig übertragbar. Um eine umsetzbare Cyclusgröße für die Peptidbibliothek zu finden, wurden Ringgrößen mit sechs bis acht Aminosäuren untersucht. Da keiner der gewählten Ringgrößen bevorzugt war, wurde die Ringgröße aus acht Aminosäuren für die Peptidbibliothek gewählt. Der Ringschluss wurde durch die Bildung einer Disulfidbrücke erreicht. Dadurch war die Alkylierung der vorhandenen Thiolgruppe in den Abbruchsequenzen notwendig. Neben den etablierten Alkylierungsreagenzien Iodessigsäure, Iodacetamid und Acrylamid wurden zwei Epoxide mituntersucht. Hierbei konnte nur bei Acrylamid und Propylenoxid eine vollständige Alkylierung beobachtet werden. Eine synthetische Peptidbibliothek aus zehn Aminosäuren pro Kopplungsschritt und einer Peptidlänge von acht Aminosäuren wurde erfolgreich nach der Split-and-Mix-Synthese hergestellt. Neben den kanonischen Aminosäuren wurde die synthetische Aminosäure 3-(3-Pyridyl)-alanin in die Peptidbibliothek mit eingebaut. Peptidsequenzen aus der cyclische Peptidbibliothek konnte mittels MALDI-TOF-MS identifiziert werden. Es konnte außerdem gezeigt werden, dass synthetische Aminosäuren mit proteinogenen Aminosäuren erfolgreich übersetzt werden. Anschließend wurde das entwickelte Screening auf eine cyclische Peptidbibliothek übertragen. Dabei konnte kein Binder identifiziert werden, da zu viele Nebenreaktionen auftraten. Ein alternativer Ringschluss über die Seitenketten von Lysin und Cystein wurden mit ortho-Phthaldialdehyd und 2,4,6-Trichloro-1,3,5-triazin (Cyanurchlorid) untersucht. Beide Bedingungen wiesen keinen erfolgreichen Ringschluss auf. Obwohl der Ringschluss über die Seitenketten von Lysin und Cystein nicht erfolgreich war, sollte ein Austausch von Cystein angestrebt werden, so dass kein freies Cystein in den Abbruchsequenzen vorhanden ist und die Alkylierung überflüssig wäre. Der Ringschluss durch Verwendung anderer Seitenketten bietet einen vielseitigen Ansatz. KW - CCMV KW - Pflanzenvirus KW - Peptidbibliothek KW - Peptid-Aptamer KW - Alkylierung KW - Cyclisierung KW - MALDI-TOF-MS KW - Fluoreszenz KW - Chip KW - Sequenzierung KW - Peptid-Synthese PY - 2022 SP - 1 EP - 81 PB - Humboldt-Universität zu Berlin CY - Berlin AN - OPUS4-54501 LA - deu AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER - TY - GEN A1 - Tchipilov, Teodor A1 - Raysyan, Anna A1 - Weller, Michael G. T1 - Methods for the quantification of particle-bound protein – Application to reagents for lateral-flow immunoassays (LFIA) T2 - Preprints N2 - Protein immobilization for the functionalization of particles is used in various applications, including biosensors, lateral-flow immunoassays (LFIA), bead-based assays, and others. Common methods for the quantification of bound protein are measuring protein in the supernatant before and after coating and calculating the difference. This popular approach has the potential for a significant overestimation of the amount of immobilized protein since layers not directly bound to the surface (soft protein corona) are usually lost during washing and handling. Only the layer directly bound to the surface (hard corona) can be used in subsequent assays. A simplified amino acid analysis method based on acidic hydrolysis and RP-HPLC-FLD of tyrosine and phenylalanine (aromatic amino acid analysis, AAAA) is proposed to directly quantify protein bound to the surface of gold nano- and latex microparticles. The results are compared with indirect methods such as colorimetric protein assays, such as Bradford, bicinchoninic acid (BCA), as well as AAAA of the supernatant. For both particle types, these indirect quantification techniques show a protein overestimation of up to 1700% compared to the direct AAAA measurements. In addition, protein coating on latex particles was performed both passively through adsorption and covalently through EDC/sulfo-NHS chemistry. Our results showed no difference between the immobilization methodologies. This finding suggests that usual protein determination methods are no unambiguous proof of a covalent conjugation on particles or beads. KW - Soft protein corona KW - Hard protein corona KW - Gold particles KW - Nanoparticles KW - Mikroparticles KW - Antibody KW - Bioconjugation KW - Protein quantification KW - Supernatant KW - Sodium chloride method KW - Covalent conjugation KW - Latex particles KW - Lateral flow immunoassays PY - 2022 UR - https://nbn-resolving.org/urn:nbn:de:kobv:b43-545365 DO - https://doi.org/10.20944/preprints202203.0332.v1 SP - 1 EP - 8 PB - MDPI CY - Basel AN - OPUS4-54536 LA - eng AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER - TY - GEN A1 - Fischer, Janina T1 - Suspensionsarray-Fluoreszenzimmunoassay zur Multiplexbestimmung von SARS-CoV-2-Antikörpern T2 - Masterarbeit N2 - Übergeordnetes Ziel der Arbeit war die Entwicklung und Optimierung eines Suspensionsarray-Fluoreszenzimmunoassays (SAFIA) für die simultane Bestimmung verschiedener gegen SARS-CoV-2-Proteine gerichtete Antikörper mittels Multiplexdetektion in komplexen Matrices wie humanen Blutserumproben. KW - SARS-CoV-2 KW - Corona KW - COVID-19 KW - Coronavirus KW - Virus KW - Spike-Protein KW - Nucleocapsid-Protein KW - RBD KW - SAFIA KW - LFIA KW - ELISA KW - Partikel KW - Mutationen KW - Neutralisierende Antikörper KW - Durchflusszytometrie KW - ACE2 PY - 2022 SP - 1 EP - 99 PB - Humboldt-Universität zu Berlin CY - Berlin AN - OPUS4-54722 LA - deu AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER - TY - GEN A1 - Ferreira Camoes Liestmann, Zoe T1 - Development of an electrochemiluminescence immunoassay for selected pathogens in wastewater T2 - Masterarbeit N2 - The outbreak of SARS-CoV-2 in December of 2019, led to a worldwide still on-going pandemic. Since then, several so-called waves of SARS-CoV-2 infections, a time period with a high and fast rising number of new infections, have occurred all over the world. Classic surveillance approaches are hardly applicable, and further, non-detected cases cannot be covered by them. Wastewater-based Epidemiology (WBE) was proven to be a reliable tool for the prediction of new SARS-CoV-2 infection waves, due to the discharge of virus particles in fecal shedding of infectious people. Until now, for the monitoring of SARS-CoV-2 in wastewater, Polymerase Chain Reaction (PCR) is used as analytical tool. Even though PCR is a highly sensitive analytical tool, is presents several disadvantages, such as the need for trained personnel, specific technical equipment, as well as a difficult performance. An analytical tool, to which these disadvantaged do not apply, are immunoassays. In this work, a sandwich Enzyme-Linked Immunosorbent Assay (ELISA), with the immobilization of the capture antibodies on the surface of a Microtiter Plate (MTP), as well as a sandwich Magnetic Bead-Based Assay (MBBA), with immobilization of the capture antibodies on the surface of Magnetic Beads (MBs), targeting the SARS-CoV-2 N-protein, were developed and optimized. Both assay formats were performed with a colorimetric and chemiluminescent detection. The developed assay is composed of the two monoclonal antibodies (mAb) AH2 and DE6 - which was biotinylated in the course of the work - which bind to two different epitops of the antigen N-protein. As tracer, Neutravidin-HRP was used, which binds, through interaction of the Neutravidin with the biotin, to the mAb DE6-Biotin. The assay development and optimization procedure included the investigation of the surface saturation with the mAb AH2, the concentration and dilution of the mAb DE6-Biotin and Neutravidin-HRP, the ideal MBs, the ideal coating as well as dilution buffers, and the colorimetric and chemiluminescent substrates. For the developed and fully optimized colorimetric ELISA, a test midpoint x0 of 388 μg/L, for the chemiluminsecent ELISA of 371 μg/L, for the colorimetric MBBA of 251 μg/L and for the chemiluminescent MBBA of 243 μg/L was obtained. Validation of the colorimetric MBBA was done by measurement of three wastewater samples collected at the Wastewater Treatment Plant (WWTP) Potsdam. Whilst no N-protein could be detected in the samples, by spiking of the wastewater samples with certain concentrations of the N-protein, 10- to 18-times lower concentrations could be back-calculated, which can be attributed to matrix-effects of the wastewater sample. Next to the matrix-effects, also several other reason exist, why no N-protein could be determined in the samples. Because of that, further investigation of the handling, and the measurement of the wastewater samples, as well as the improvement of the assay sensitivity through further optimization steps or exchange of the antibodies, is still necessary. KW - SARS-CoV-2 KW - ELISA KW - Antibody KW - N-capsid PY - 2022 SP - 1 EP - 102 PB - Technischen Universität München CY - München AN - OPUS4-57744 LA - eng AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER - TY - GEN A1 - von der Au, Marcus A1 - Meermann, Björn T1 - Dem Leben auf der Spur – Einzelzellanalytik T2 - Faszination Chemie N2 - Zellen, als Grundeinheit für Organismen, bilden einen wesentlichen Eckpfeiler für das Leben. Die Untersuchung von einzelnen Zellen liefert wertvolle Einblicke in fundamentale Prozesse des Lebens. So können Anomalien auf der Zelleebene Indikatoren für Krankheiten sein und durch eine Analyse früh erkannt werden. Weiterhin kann durch ein tiefgreifendes Verständnis von Vorgängen in Zellen auch gezielt Forschung zu z.B. neuen Medikamenten betrieben werden. Damit können die Wirksamkeit erhöht und die Nebenwirkungen reduziert werden. KW - SC-ICP-MS KW - Algen KW - Cispaltin PY - 2021 UR - https://faszinationchemie.de/wissen-und-fakten/news/dem-leben-auf-der-spur-einzelzellanalytik/ SP - 1 EP - 4 AN - OPUS4-53859 LA - deu AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER - TY - GEN A1 - Wilke, Marco A1 - Röder, Bettina A1 - Paul, Martin A1 - Weller, Michael G. T1 - Sintered Glass Monoliths as New Supports for Affinity Columns T2 - Preprints N2 - A novel stationary phase for affinity separations is presented. This material is based on sintered borosilicate glass readily available as semi-finished filter plates with defined porosity and surface area. The material shows fast binding kinetics and excellent long-term stability under real application conditions due to lacking macropores and high mechanical rigidity. The glass surface can be easily modified with standard organosilane chemistry to immobilize selective binders or other molecules used for biointeraction. In this paper, the manufacturing of the columns and their respective column holders by 3D printing is shown in detail. The model system protein A/IgG was chosen as an example to examine the properties of such monolithic columns under realistic application conditions. Several specifications, such as (dynamic) IgG capacity, pressure stability, long-term performance, productivity, non-specific binding, and peak shape, are presented. It could be shown that due to the very high separation speed, 250 mg antibody per hour and column can be collected, which surpasses the productivity of most standard columns of the same size. The total IgG capacity of the shown columns is around 4 mg (5.5 mg/mL), which is sufficient for most tasks in research laboratories. The cycle time of an IgG separation can be less than 1 minute. Due to the glass material's excellent pressure resistance, these columns are compatible with standard HPLC systems. This is usually not the case with standard affinity columns, limited to manual use or application in low-pressure systems. The use of a standard HPLC system also improves the ability for automation, which enables the purification of hundreds of cell supernatants in one day. The sharp peak shape of the elution leads to an enrichment effect, which might increase the concentration of IgG by a factor of 3. The final concentration of IgG can be around 7.5 mg/mL without the need for an additional nanofiltration step. The purity of the IgG was > 95% in one step and nearly 99% with a second polishing run. KW - Affinity Chromatography KW - Glass KW - Purification KW - Antibodies KW - Solid support KW - HPLC KW - FPLC KW - Separation PY - 2021 UR - https://nbn-resolving.org/urn:nbn:de:kobv:b43-529117 DO - https://doi.org/10.20944/preprints202103.0298.v1 SP - 1 PB - MDPI CY - Basel AN - OPUS4-52911 LA - eng AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER - TY - GEN A1 - Iwert, C. A1 - Stein, J. A1 - Appelt, C. A1 - Vogt, K. A1 - Rainer, R. J. A1 - Tummler, K. A1 - Mühle, K. A1 - Stanko, K. A1 - Schumann, J. A1 - Uebe, D. A1 - Jürchott, K. A1 - Lisec, Jan A1 - Janek, K. A1 - Gille, C. A1 - Textoris-Taube, K. A1 - Sai, S. A1 - Petersen, A. A1 - Kühl, A. A. A1 - Klipp, E. A1 - Meisel, C. A1 - Sawitzki, B. T1 - TCAIM controls effector T cell generation by preventing Mitochondria-Endoplasmic Reticulum Contact Site-initiated Cholesterol Biosynthesis T2 - bioRxiv N2 - T cells need to adapt their cellular metabolism for effector cell differentiation. This relies on alterations in mitochondrial physiology. Which signals and molecules regulate those alterations remains unclear. We recently reported, that the mitochondrial protein TCAIM inhibits activation-induced changes in mitochondrial morphology and function and thus, CD effector T cell formation. Using conditional TCAIM knock-in (KI) and knockout (KO) mice, w now show that it also applies to CD8+ T cells and more importantly, delineate the molecular processes in mitochondria by which TCAIM controls effector cell differentiation. TCAIM KI resulted in reduced activation-induced HIF1α protein expression. Metabolomics and transcriptional data in combination with mathematical flux modeling revealed an impaired induction of anabolic pathways, especially of the mevalonate pathway and cholesterol biosynthesis in TCAIM KI CD8+ T cells. Addition of cholesterol completely rescued HIF1α protein expression, activation and proliferation of TCAIM KI CD8+ T cells. At the molecular level, TCAIM delayed activation-induced mitochondria-ER contact (MERC) formation by binding to MERC promoting proteins such as RMD3 and VDAC2. In summary, we demonstrate that TCAIM suppresses effector cell differentiation by inhibiting MERC formation, which induce HIF1α-mediated increase in cellular metabolism and cholesterol biosynthesis. KW - Mass Spectroscopy KW - Metabolomics KW - Cancer PY - 2021 UR - https://nbn-resolving.org/urn:nbn:de:kobv:b43-533543 UR - https://www.biorxiv.org/content/10.1101/2021.04.20.440500v1 DO - https://doi.org/10.1101/2021.04.20.440500 VL - April SP - 1 EP - 45 PB - Cold Spring Harbor Laboratory AN - OPUS4-53354 LA - eng AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER - TY - GEN A1 - Paul, Martin A1 - Tannenberg, Robert A1 - Tscheuschner, Georg A1 - Wilke, Marco A1 - Weller, Michael G. T1 - Cocaine Detection by a Laser-induced Immunofluorometric Biosensor T2 - Preprints N2 - The trafficking of illegal drugs by criminal networks at borders, harbors, or airports is an increasing issue in public health as these routes ensure the main supply of illegal drugs. The prevention of drug smuggling, including the installation of scanners and other analytical devices to detect ultra-small traces of drugs within a reasonable time frame, remains a challenge. The presented immunosensor is based on a monolithic affinity column with a large excess of immobilized hapten, which traps fluorescently labeled antibodies as long as the analyte cocaine is absent. In the presence of the drug, some binding sites of the antibody will be blocked, which leads to an immediate breakthrough of the labeled protein, detectable by highly sensitive laser-induced fluorescence with the help of a Peltier-cooled complementary metal-oxide-semiconductor (CMOS) camera. Liquid handling is performed with high-precision syringe pumps and microfluidic chip-based mixing devices and flow cells. The biosensor achieved limits of detection of 23 pM (7 ppt) of cocaine with a response time of 90 seconds and a total assay time below 3 minutes. With surface wipe sampling, the biosensor was able to detect 300 pg of cocaine. This immunosensor belongs to the most sensitive and fastest detectors for cocaine and offers near-continuous analyte measurement. KW - Drug search KW - Customs KW - Confiscation KW - Border surveillance KW - Narcotics KW - International drug trade KW - Drug trafficking KW - Illicit drug KW - Immunosensor KW - Antibodies KW - Detection PY - 2021 UR - https://nbn-resolving.org/urn:nbn:de:kobv:b43-529951 DO - https://doi.org/10.20944/preprints202107.0521.v1 SP - 1 PB - MDPI CY - Basel AN - OPUS4-52995 LA - eng AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER - TY - GEN A1 - Döring, Sarah T1 - Vergleichende Darstellung SARS-CoV-2-spezifischer Nanobodys aus unterschiedlichen Wirtsorganismen T2 - Masterarbeit N2 - Aufgrund der anhaltenden COVID-19-Pandemie werden neutralisierende Therapeutika benötigt. Eine Möglichkeit zur Behandlung stellt die Verwendung monoklonaler Anti-SARS-CoV-2-Immun-globuline dar. Ihre Produktion in Säugetierzellen ist jedoch schwer skalierbar, um den weltweiten Bedarf zu decken. VHH-Antikörper, auch Nanobodys genannt, bieten hierfür eine Alternative, da sie eine hohe Temperaturstabilität aufweisen und eine kostengünstige Produktion in prokaryotischen Wirtsorganismen ermöglichen. KW - E. coli KW - Corona KW - Virus KW - Spike-Protein KW - Nanobody KW - Antikörper KW - Expression KW - Fingerprint KW - Vhh KW - RBD KW - COVID-19 KW - SARS-CoV-2 KW - ELISA KW - MST KW - Halomonas elongata KW - Periplasma KW - SDS-PAGE KW - ACE2-Rezeptor PY - 2021 SP - 1 EP - 111 PB - Technische Universität Berlin CY - Berlin AN - OPUS4-54624 LA - deu AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER - TY - GEN A1 - López de Ipina, J.-M. A1 - Arevalillo, A. A1 - Martín, A. A1 - Caillard, B. A1 - Marcoulaki, E. A1 - Aguerre- Charol, O. A1 - van Duuren-Stuurman, B. A1 - Hodoroaba, Vasile-Dan A1 - Viitanen, A.-K. A1 - Witters, H. A1 - Vercauteren, S. A1 - Persson, K. A1 - Bard, D. A1 - Evans, G. A1 - Jensen, K.A. A1 - Himly, M. A1 - Scalbi, S. A1 - Papin, A. A1 - Le Bihan, O. A1 - Kanerva, T. A1 - Tirez, K. A1 - Frijns, E. A1 - Niga, P. A1 - Eleftheriadis, K. A1 - Travlos, A. A1 - Geppert, M. A1 - Himly, M. A1 - Radnik, Jörg A1 - Kuchenbecker, Petra A1 - Resch-Genger, Ute A1 - Fraboulet, I. A1 - Bressot, C. A1 - Rissler, J. A1 - Gaucher, R. A1 - Binotto, G. A1 - Krietsch, Arne A1 - Braun, A. A1 - Abenet, S. A1 - Catalan, J. A1 - Verstraelen, S. A1 - Manier, N. A1 - Manzo, S. A1 - Fransman, S. A1 - Queron, J. A1 - Charpentier, D. A1 - Taxell, D. A1 - Säämänen, A. A1 - Brignon, J.-M. A1 - Jovanovic, A. A1 - Bisson, M A1 - Neofytou, P. T1 - EC4Safenano - Catalogue of Services N2 - The publicly available document encapsulates the first version of the Catalogue of Services of the future EC4Safenano Centre (CoS 2019). The CoS 2019 is structured in 12 Service Categories and 27 Service Topics, for each of the 12 categories considered. This architecture configures a 12 x 27 matrix that allows ordering the potential EC4Safenano offer in 324 types of services/groups of services. Each type of service/group of services is described, in a simple and friendly way, by means of a specific service sheet: the EC4Safenano - Service Data Sheet (EC4-SDS). These EC4-SDSs allow structuring and summarizing the information of each service, providing the customer with a concise view of characteristics of the service and also the contact details with the service provider. The CoS 2019 deploys a map of services consisting of a set of 100 EC4-SDSs, covering 7 of the 12 Service Categories and 17 of the 27 Service Topics. The harmonization of services is visualized as a future necessary step in EC4Safenano, in order to strengthen the offer and provide added value to customers with a growing offer of harmonized services in future versions of the CoS. The information contained in this document is structured in 3 main sections, as follows: • Catalogue structure. This section describes in short the main characteristics of the CoS 2019. • Catalogue content. This section represents the core part of the document and encapsulates the set of 100 SDSs displaying the offer proposed by the CoS 2019. • Online Catalogue. This section describes the resources implemented by EC4Safenano to facilitate the on-line consultation of the CoS 2019 by customers and other interested parties. KW - Nano-safety KW - Analytical services KW - Nanomaterials KW - Catalogue of services KW - EC4SafeNano KW - European Centre PY - 2021 UR - https://ec4safenano.eu-vri.eu/Public/Guidance SP - 1 EP - 72 PB - EU-VRi – European Virtual Institute for Integrated Risk Management CY - Stuttgart, Germany AN - OPUS4-52943 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 - Herrmann, Stefan T1 - Charakterisierung zweier monoklonaler Antikörper zur Detektion von Sprengstoffen T2 - Masterarbeit N2 - In 2019 over 30 000 people were killed or injured by explosions caused by explosives like TNT, PETN, HMX and RDX. Therefore, highly sensitive assays for the detection of TNT are needed. In this study we compared two commercially available TNT antibodies: A1.1.1 and EW75C with a highly optimized indirect competitive ELISA based on a BSA-TNA conjugate. As a result, a precision profile for both antibodies was determined with a LOD of 170 pmol L-1 for the clone A1.1.1 and a LOD of 3,2 nmol L-1 for the clone EW75C. The measurements showed that the clone A1.1.1 is a highly sensitive antibody for the detection of TNT while the clone EW75C does show medium performance at most. In the cross-reactivity characterization of both antibodies many substances, closely related to the structure of TNT were tested. Both antibodies showed strong cross reactivity with trinitroaniline and trinitrobenzene. For the clone A1.1.1, which is known to originate from immunization of mice with an TNP-glycine-KLH conjugate, this has to be expected. Interestingly the clone EW75C, which was not characterized yet, showed similar behavior. This suggests a TNA-conjugate as immunogen for the EW75C antibody as well. None of both antibodies showed cross-reactions to the high explosives PETN, HMX and RDX. Also, the cross-reactions of nitro musks with the antibodies were investigated. Despite their prohibition, nitro musks are still used in Asia especially and are particularly popular in India. The overall superior clone A1.1.1 showed a significant cross-reactivity to musk ambrette. For practical reasons the influence of musk ambrette on this assay when used in natural environment should be investigated. In further experiments, the highly sensitive TNT antibody A1.1.1 was digested with papain to obtain monovalent Fab-fragments. Due to its high stability against the digestion, a custom protocol for the IgG1 subclass of mice, to which the clone A1.1.1 belongs, was developed, resulting in a quantitative digestion of the intact antibody to Fab fragments. The success of the digestion was determined with MALDI-TOF-MS and SDS-PAGE. It was shown that this protocol worked for many different antibodies of IgG1 subclass as well. KW - TNT KW - Trinitrotoluol KW - Nitroaromaten KW - Nitromoschus KW - Duftstoffe KW - ELISA KW - Immunoassay KW - Antibody KW - Explosives KW - Klon A1.1.1 KW - Klon EW75C KW - Fab Fragment KW - Kreuzreaktion KW - Crossreactivity KW - Precision Profile KW - Präzisionsprofil KW - Affinity KW - Affinitätskonstante KW - IC50 PY - 2020 SP - 1 EP - 113 PB - Hochschule für Technik und Wirtschaft Berlin - htw CY - Berlin AN - OPUS4-54556 LA - deu AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER - TY - GEN A1 - Luco Colilles, Arnau T1 - Evaluation of two compact near-infrared spectrometers for the detection of microplastics in soil samples T2 - Final Degree Projects (TFG) - Chemistry N2 - In the last years, the Process Analytical Technology (PAT) research field has become of great importance due to its connection with the industrial world. This industry related research, along with the evolutionary progress that technology has seen lately, has allowed that key analytical techniques in an industrial environment have undergone a radical development. One of these techniques has been the near-infrared spectroscopy (NIRS). This technique, currently common in "online" industrial analysis, has seen a remarkable revolution, especially since the introduction of the microelectromechanical systems technology (MEMS) in the spectroscopy field. Nowadays it is possible to find compact spectrometers no bigger than a wristwatch in the market. Although there is an unavoidable question: can these compact spectrometers actually compete against the traditional spectrometers? In this project different calibration parameters of two compact NIR spectrometers ("Spectral Engines Oy NIR-One Sensor NM2.0", with 1550 to 1950 nm range; and "NeoSpectra Si-ware", with 1300 to 2550 nm range) were evaluated, and the obtained results were compared with a reference spectrometer ("Bruker Optik GmbH Matrix-F", with 15000 to 4000 cm–1 range). In order to obtain the different calibration parameters, a sequence of quality performance tests were conducted. The results obtained after the different experiments carried out with both compact spectrometers prove that their performance is more than acceptable for routine analysis. Afterward, model samples of different microplastics in soil at different known concentration were analyzed with all three spectrometers. Chemometric models capable to identify and classify microplastics in soil were established. For this analysis five of the most used plastics worldwide were used: polyethylene (PE), polyethylene terephthalate (PET), polypropylene (PP), polystyrene (PS), and polyvinyl chloride (PVC). After the Principal component analysis (PCA), it can be seen that only the NIR-One NM2.0 is capable to differentiate all types of microplastics in soil at concentrations of 1–2 %, while the NeoSpectra Si-ware is unable to identify the PET sample. KW - Near-infrared KW - Microplastics KW - Compact spectrometers PY - 2019 UR - https://nbn-resolving.org/urn:nbn:de:kobv:b43-511626 UR - http://hdl.handle.net/2445/138198 SP - 1 EP - 29 PB - Universitat de Barcelona CY - Barcelona AN - OPUS4-51162 LA - eng AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER - TY - GEN A1 - Paul, Martin A1 - Weller, Michael G. T1 - Antibody Screening by Microarray Technology – Direct Identification of Selective High-Affinity Clones T2 - Preprints N2 - The primary screening of hybridoma cells is a time-critical and laborious step during the development of monoclonal antibodies. Often critical errors occur in this phase, which supports the notion that the generation of monoclonal antibodies with hybridoma technology is difficult to control and hence a risky venture. We think that it is crucial to improve the screening process to eliminate most of the immanent deficits of the conventional approach. With this new microarray-based procedure, several advances could be achieved: Selectivity for excellent binders, high throughput, reproducible signals, avoidance of misleading avidity (multivalency) effects, and simultaneous performance of competition experiments. The latter can directly be used to select clones of desired cross-reactivity properties. In this paper, a model system with two excellent clones against carbamazepine, two weak clones and blank supernatant has been designed to examine the effectiveness of the new system. The excellent clones could be detected largely independent of the IgG concentration, which is unknown during the clone screening since the determination and subsequent adjustment of the antibody concentration is not possible in most cases. Furthermore, in this approach, the enrichment, isolation, and purification of IgG for characterization is not necessary. Raw cell culture supernatant can be used directly, even when fetal calf serum (FCS) or other complex media had been used. In addition, an improved method for the oriented antibody-immobilization on epoxy-silanized slides is presented. Based on the results of this model system, we conclude that this approach should be preferable to most other protocols leading to many of false positives, causing expensive and lengthy confirmation steps to weed out the poor clones. KW - Hybridoma KW - Monoclonal Antibodies KW - Clones KW - Competitive Immunoassay KW - Hapten Immunoassay KW - False Positives PY - 2019 UR - https://nbn-resolving.org/urn:nbn:de:kobv:b43-506621 DO - https://doi.org/10.20944/preprints201911.0023.v1 SN - 2310-287X SP - 1 EP - 17 PB - MDPI CY - Basel AN - OPUS4-50662 LA - eng AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER - TY - GEN A1 - Tscheuschner, Georg T1 - Entwicklung einer Methode zur schnellen Identifikation von Antikörpern mittels MALDI-TOF-MS T2 - Masterarbeit N2 - Im Vergleich zu anderen Proteinen ist die Identifizierung von Antikörpern anhand ihrer Sequenz zum Beispiel mittels "peptide mass fingerprinting" schwierig. Da die Sequenzinformation eines Antikörpers aufgrund der hypersomatischen Mutation während der Affinitätsreifung nicht im Genom eines Organismus gespeichert ist, kann die Aminosäuresequenz nicht auf einfachem Weg der DNA-Sequenzierung gewonnen werden. Das ist nur in seltenen Fällen möglich, wenn dem Endanwender der Zellklon der Antikörper-produzierenden Zelle zugänglich ist. Eine Sequenzierung auf Protein-Ebene ist sehr aufwändig und teuer und wird daher fast nie für die Charakterisierung von analytischen Antikörpern verwendet. Der Mangel an Validierung dieser analytischen Antikörper, die bei Experimenten verwendeten werden, löst aber eine Reihe Probleme aus, die die Wiederholbarkeit dieser Experimente schwierig und in einigen Fällen unmöglich macht. Das sorgt jährlich für verschwendete Forschungsgelder in Milliardenhöhe und hindert den wissenschaftlichen Fortschritt. Ziel der vorliegenden Arbeit war die Entwicklung einer einfachen und schnellen Methode, die es trotzdem ermöglicht, die Identifikation von Antikörpern sicherzustellen. Dazu wurde eine Methode basierend auf dem "peptide mass fingerprinting" gewählt. Das Problem der unbekannten Aminosäuresequenz der Antikörper wurde gelöst, indem lediglich die Peptidmuster der entstehenden Fingerprint-Spektren zur Identifikation herangezogen wurden. MALDI wurde dabei als Ionisationsmethode für die Massenspektrometrie gewählt, da die resultierenden Spektren im Gegensatz zu ESI-MS einfach auszuwerten sind. Auch kann auf eine vorige Trennung der Peptide mittels LC verzichtet werden, was zusätzlich Analysenzeit spart. Für die Proteinspaltung wurde eine simple saure Hydrolyse mittels Ameisensäure gewählt. Im Vergleich zum herkömmlichen Trypsin-Verdau konnten auf zeitraubende Arbeitsschritte wie Denaturierung, Reduktion und Alkylierung der Antikörper verzichtet werden. Die Hydrolyse mittels Ameisensäure wurde bisher nur auf kleine und mittelgroße Proteine angewendet, sodass im ersten Teil dieser Arbeit mehrere Schritte optimiert wurden bevor zufriedenstellende Fingerprint-Spektren von Antikörpern erhalten wurden. KW - Peptide KW - Fingerprint KW - Peptide mass fingerprinting KW - Massenspektrometrie KW - Saure Hydrolyse KW - Festphasenextraktion KW - Protein G KW - Ameisensäure KW - ABID KW - Korrelationsmatrix PY - 2019 SP - 1 EP - 116 PB - Humboldt-Universität zu Berlin CY - Berlin AN - OPUS4-54626 LA - deu AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER - TY - GEN A1 - Schwaar, Timm A1 - Remmler, Dario A1 - Börner, Hans G. T1 - Spec2Seq T2 - edoc-Server: Open-Access-Publikationsserver der Humboldt-Universität N2 - Spe2Seq automatically translates mass spectrometry fragment spectra of precision polymers into their corresponding sequence. This software is especially useful for combinatorial approaches. KW - Java GUI KW - Polymers KW - Mass spectroscopy (MS) PY - 2018 SP - 1 EP - 8 PB - Humboldt-Universität CY - Berlin AN - OPUS4-46987 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 - TY - GEN A1 - Sauer, Andreas A1 - Buttler, Sabine A1 - Becker, Roland T1 - Bericht zum 24. BAM-Ringversuch "Altlasten" N2 - Präsentation der Ergebnisse eines Ringversuchs zur Kompetenzbewertung von Prüflaboratorien auf dem Gebiet der anorganischen und organischen Bodenanalytik: Polychlorierte Biphenyle (PCB) in Boden, Mineralölkohlenwasserstoffe (MKW) in Boden, Elemente in Boden und Gesamtcyanid in Boden. KW - Spurenelemente KW - Boden KW - Gesamtcyanid KW - Eignungsprüfung KW - MKW KW - PCB PY - 2018 UR - https://nbn-resolving.org/urn:nbn:de:kobv:b43-473418 SP - 1 EP - 183 PB - Bundesanstalt für Materialforschung und -prüfung (BAM) CY - Berlin AN - OPUS4-47341 LA - deu AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER - TY - GEN A1 - Völzke, Jule Lexa T1 - Herstellung und Charakterisierung Antigen-beschichteter Nanopartikel als Analoga von Virus-like Particles (VLP) T2 - Masterarbeit N2 - In dieser Arbeit wurden drei verschiedene Nanomaterialien auf ihre Bindungsfähigkeit zu Proteinen untersucht. Zu Beginn standen dabei die Herstellung stabiler Dispersionen der einzelnen Nanopartikel und die Stabilität der gebildeten Konjugate im Vordergrund. Der Nachweis einer erfolgreichen Konjugatbildung, sprich der Beschichtung von Nanopartikel mit Proteinen, wurde sowohl qualitativ mittels DLS-Messungen als auch über quantitative Protein-Bestimmungen erbracht. Für die Quantifizierung konnten verschiedene Methoden eingesetzt werden. Neben der klassischen Vorgehensweise, welche indirekt über die Quantifizierung von ungebundenem Protein im Überstand erfolgt, konnten ihm Rahmen dieser Arbeit verschiedene direkte Bestimmungsmethoden entwickelt werden. So wurden mittels kolorimetrischer Tests, wie dem BCA-Assay und dem Bradford-Assay, Nanodiamantdispersionen mit Hilfe einer Korrekturwellenlänge vermessen und quantifiziert. Ebenso zum Einsatz kam die Methode der Aminosäureanalytik, welche aufgrund ihrer guten Rückführbarkeit auf Aminosäurestandards Ergebnisse mit hoher Richtigkeit generieren kann und ebenso die Detektion kleiner Proteinmengen möglich macht. Nach den erfolgten quantitativen Betrachtungen wurden die Protein-beschichteten Nanopartikel auf ihre Anwendbarkeit als Analoga von Virus-like Particles (VLP) bei einer Immunisierung zur Gewinnung von polyklonalen Antikörpern gegen humanes Ceruloplasmin in Kaninchen überprüft. Es konnte mittels ELISA gezeigt werden, dass die Konjugate erfolgreich für die Herstellung von Antikörpern eingesetzt werden können und im zeitlichen Verlauf einer Immunisierung eine Steigerung des Antikörper-Titers zu erreichen ist. KW - DLS KW - Dynamische Lichtstreuung KW - Aluminiumoxid KW - Nanodiamant KW - Gold-Nanopartikel KW - BSA KW - Albumin KW - Protein G KW - Ceruloplasmin KW - Immunpräzipitation KW - Ultraschall KW - BCA KW - Bradford-Assay KW - AAAA KW - Aromatische Aminosäureanalytik KW - ICP-MS KW - NaCl-Methode PY - 2018 SP - 1 EP - 107 PB - Humboldt-Universität zu Berlin CY - Berlin AN - OPUS4-54625 LA - deu AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER -