TY - JOUR A1 - Tannenberg, Robert A1 - Paul, Martin A1 - Röder, Bettina A1 - Gande, S. L. A1 - Sreeramulu, S. A1 - Saxena, K. A1 - Richter, C. A1 - Schwalbe, H. A1 - Swart, C. A1 - Weller, Michael G. T1 - Chemiluminescence biosensor for the determination of cardiac troponin I (cTnI) N2 - Cardiac troponin I (cTnI) is a crucial biomarker for diagnosing cardiac vascular diseases, including acute myocardial infarction (AMI). This study presents a proof-of-concept chemiluminescence-based immunosensor for rapid and accurate measurement of cTnI, with the potential for online monitoring. The immunosensor incorporates a flow cell design and a sensitive complementary metal-oxide-semiconductor (CMOS) camera for optical readout. A microfluidic setup was established to enable selective and quasi-online determination of cTnI within ten minutes. The sensor was tested with recombinant cTnI in phosphate buffer, demonstrating measurements in the concentration range of 2–25 µg/L, with a limit of detection (LoD) of 0.6 µg/L (23 pmol/L) achieved using the optimized system. The immunosensor exhibited high selectivity, as no cross-reactivity was observed with other recombinant proteins such as cTnT and cTnC at a concentration of 16 µg/L. Measurements with diluted blood plasma and serum yielded an LoD of 60 µg/L (2.4 nmol/L) and 70 µg/L (2.9 nmol/L), respectively. This biosensor offers a promising approach for the rapid and sensitive detection of cTnI, contributing to the diagnosis and management of acute myocardial infarction and other cardiac vascular diseases. N2 - Das kardiale Troponin I (cTnI) ist ein wichtiger Biomarker für die Diagnose von Herz-Kreislauf-Erkrankungen, einschließlich des akuten Myokardinfarkts (AMI). In dieser Studie wird ein auf Chemilumineszenz basierender Immunsensor für die schnelle und genaue Messung von cTnI vorgestellt, der das Potenzial für eine Online-Überwachung hat. Der Immunsensor besteht aus einer Durchflusszelle und einer empfindlichen CMOS-Kamera (Complementary Metal-Oxide-Semiconductor) zur optischen Detektion. Es wurde ein mikrofluidischer Aufbau entwickelt, der eine selektive und quasi Online-Bestimmung von cTnI innerhalb von zehn Minuten ermöglicht. Der Sensor wurde mit rekombinantem cTnI in Phosphatpuffer getestet und zeigte einen Messbereich von 2-25 µg/L, wobei mit dem optimierten System eine Nachweisgrenze (LoD) von 0,6 µg/L (23 pmol/L) erreicht wurde. Der Immunsensor zeigte eine hohe Selektivität, da keine Kreuzreaktivität mit anderen rekombinanten Proteinen wie cTnT und cTnC bei einer Konzentration von 16 µg/L beobachtet wurde. Messungen mit verdünntem Blutplasma und Serum ergaben einen LoD von 60 µg/L (2,4 nmol/L) bzw. 70 µg/L (2,9 nmol/L). Dieser Biosensor bietet einen vielversprechenden Ansatz für den schnellen und empfindlichen Nachweis von cTnI, der zur Diagnose und Behandlung des akuten Myokardinfarkts und anderer kardialer Gefäßerkrankungen beitragen kann. KW - Acute myocardial infarction KW - Heart attack KW - Emergency KW - Diagnosis KW - Cardiac troponin KW - Biomarker KW - Immunosensor KW - Biosensor KW - Chemiluminescence KW - Luminol KW - Peroxidase KW - Monoclonal antibodies KW - Flow injection immunoassay KW - Immunometric assay KW - Immunometric biosensor KW - Microfluidic system KW - Monolithic column KW - Online biosensor PY - 2023 UR - https://nbn-resolving.org/urn:nbn:de:kobv:b43-575471 DO - https://doi.org/10.3390/bios13040455 SN - 2079-6374 VL - 13 IS - 4 SP - 1 EP - 20 PB - MDPI CY - Basel AN - OPUS4-57547 LA - eng AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER - TY - JOUR A1 - Völzke, Jule L. A1 - Smatty, Sarah A1 - Döring, Sarah A1 - Ewald, Shireen A1 - Oelze, Marcus A1 - Fratzke, Franziska A1 - Flemig, Sabine A1 - Konthur, Zoltán A1 - Weller, Michael G. T1 - Efficient Purification of Polyhistidine-Tagged Recombinant Proteins Using Functionalized Corundum Particles N2 - Immobilized metal affinity chromatography (IMAC) is a widely used technique for purifying polyhistidine-tagged recombinant proteins. However, it often has practical limitations that require complex optimizations and additional steps for purification. In this study, we introduce functionalized corundum particles as a novel, efficient, and economical method for purifying recombinant proteins in a column-free format. The corundum surface is modified with amino silane APTES, followed by EDTA dianhydride, and then loaded with nickel ions. We used the Kaiser test to monitor the modification process and ICP-MS to quantify the metal-binding capacity. To evaluate the system, we used His-tagged protein A/G (PAG) mixed with bovine serum albumin (BSA). The corundum particles exhibited a binding capacity of approximately 3 mg of protein per gram of corundum or 2.4 mg per 1 mL of corundum suspension. We also examined cytoplasm obtained from different E. coli strains as an example of a complex matrix. Varying the imidazole concentration in the loading and washing buffers showed that higher concentrations during loading improved purity. Even with sample volumes as large as one liter, we successfully isolated recombinant proteins down to a concentration of 1 µg/mL. We found higher purity levels with corundum when comparing the corundum material to standard Ni–NTA agarose beads. We successfully purified His6-MBP-mSA2, a fusion protein comprising monomeric streptavidin and maltose-binding protein, from E. coli cytoplasm, demonstrating the method's applicability. We also purified SARS-CoV-2-S-RBD-His8 expressed in human Expi293F cells, confirming its suitability for mammalian cell culture supernatants. The material cost of the nickel-loaded corundum material (without regeneration) is estimated to be less than 30 cents per gram of functionalized support or 10 cents per milligram of isolated protein. Another advantage of this system is the exceptional physical and chemical stability of corundum particles. Overall, we have demonstrated that this novel material offers an efficient, robust, and cost-effective purification platform for His-tagged proteins, even in challenging, complex matrices and large sample volumes with low product concentrations. This method has potential applications in both small laboratories and large-scale industrial settings. N2 - Die immobilisierte Metallaffinitätschromatographie (IMAC) ist eine weit verbreitete Technik zur Reinigung von rekombinanten Proteinen mit Polyhistidin-Markierung. Sie hat jedoch oft praktische Einschränkungen, die komplexe Optimierungen und zusätzliche Schritte für die Aufreinigung erfordern. In dieser Studie stellen wir funktionalisierte Korundpartikel als neuartige, effiziente und wirtschaftliche Methode zur Reinigung rekombinanter Proteine in einem säulenfreien Format vor. Die Korundoberfläche wird mit dem Aminosilan APTES und anschließend mit EDTA-Dianhydrid modifiziert und dann mit Nickelionen beladen. Wir haben den Kaiser-Test zur Überwachung des Modifizierungsprozesses und ICP-MS zur Quantifizierung der Metallbindungskapazität verwendet. Zur Charakterisierung des Systems verwendeten wir His-markiertes Protein A/G (PAG) in Kombination mit Rinderserumalbumin (BSA). Die Korundpartikel wiesen eine Bindungskapazität von etwa 3 mg Protein pro Gramm Korund oder 2,4 mg pro 1 ml Korundsuspension auf. Als Beispiel für eine komplexe Matrix untersuchten wir auch Zytoplasma, das aus verschiedenen E. coli-Stämmen gewonnen wurde. Die Variation der Imidazolkonzentration in den Lade- und Waschpuffern zeigte, dass höhere Konzentrationen beim Laden die Reinheit verbesserten. Selbst bei einem Probenvolumen von bis zu einem Liter konnten wir rekombinante Proteine von 1 µg/mL isolieren. Beim Vergleich des Korundmaterials mit Standard-Ni-NTA-Agarose-Beads stellten wir einen höheren Reinheitsgrad mit Korund fest. Wir reinigten erfolgreich His6-MBP-mSA2 aus E. coli-Zytoplasma, ein Fusionsprotein, das aus monomerem Streptavidin und Maltose-bindendem Protein besteht. Wir reinigten auch SARS-CoV-2-S-RBD-His8, das in humanen Expi293F-Zellen exprimiert wurde, und bestätigten damit die Eignung des Materials für Zellkulturüberstände von Säugetieren. Die Materialkosten für nickelbeladenen Korund (ohne Regenerierung) werden auf weniger als 30 Cent pro Gramm funktionalisierten Trägers oder 10 Cent pro Milligramm isolierten Proteins geschätzt. Ein weiterer Vorteil dieses Systems ist die außergewöhnliche physikalische und chemische Stabilität der Korundpartikel. Insgesamt haben wir gezeigt, dass dieses neuartige Material eine effiziente, robuste und kostengünstige Reinigungsplattform für His-markierte Proteine bietet, selbst bei schwierigen, komplexen Matrices und großen Probenmengen mit niedrigen Produktkonzentrationen. Diese Methode könnte sowohl in kleinen Labors als auch in der Großindustrie eingesetzt werden. KW - Aluminum oxide KW - Sapphire KW - Ethylenediaminetetraacetic acid KW - Nickel chelate KW - EDTAD KW - HexaHis-Tag KW - His6 KW - 6xHis KW - His8 KW - Bioseparation KW - IMAC purification KW - Immunocapture KW - Affinity chromatography KW - Carrier KW - Nickel KW - Recombinant protein KW - Escherichia coli KW - Bacterial lysates KW - Cytoplasm KW - Polishing KW - Downstream processing PY - 2023 UR - https://nbn-resolving.org/urn:nbn:de:kobv:b43-575672 DO - https://doi.org/10.3390/biotech12020031 VL - 12 IS - 2 SP - 1 EP - 18 PB - MDPI CY - Basel, Schweiz AN - OPUS4-57567 LA - eng AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER - TY - JOUR A1 - Paul, Martin A1 - Weller, Michael G. T1 - Antibody screening by microarray technology - Direct identification of selective high-affinity clones 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 critical 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 performance of simultaneous competition experiments. The latter can also 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 containing fetal bovine serum was designed to examine the effectiveness of the new system. The excellent clones could be detected largely independent of the immunoglobulin G (IgG) concentration, which is usually unknown during the clone screening since the determination and subsequent adjustment of the antibody concentration are not feasible 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 is 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 with simulated hybridoma supernatants, we conclude that this approach should be preferable to most other protocols leading to many false positives, causing expensive and lengthy elimination steps to weed out the poor clones. KW - ELISA KW - Immunoassay KW - Microarray KW - Lab-on-a-chip KW - Miniaturization KW - Aautomatisation KW - HTS KW - High-throughput KW - Screening KW - Fluorescence KW - Label KW - Hybridoma KW - Inhibition PY - 2020 UR - https://nbn-resolving.org/urn:nbn:de:kobv:b43-503361 DO - https://doi.org/10.3390/antib9010001 SN - 2073-4468 VL - 9 IS - 1 SP - 1 EP - 16 PB - MDPI CY - Basel AN - OPUS4-50336 LA - eng AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER - TY - JOUR A1 - Döring, Sarah A1 - Tscheuschner, Georg A1 - Flemig, Sabine A1 - Weller, Michael G. A1 - Konthur, Zoltán T1 - Cost-Effective Method for Full-Length Sequencing of Monoclonal Antibodies from Hybridoma Cells N2 - Background: Monoclonal antibodies play an important role in therapeutic and analytical applications. For recombinant expression, the coding sequences of the variable regions of the heavy and light chains are required. In addition, cloning antibody sequences, including constant regions, reduces the impact of hybridoma cell loss and ensures preservation of the naturally occurring full antibody sequence. Method: We combined amplification of IgG antibody variable regions from hybridoma mRNA with an advanced method for fulllength cloning of monoclonal antibodies in a simple two-step workflow. Following Sanger sequencing and evaluation of consensus sequences, the best matching variable, diversity, and joining (V-(D-)J) gene segments were identified according to identity scores from IgBLAST reference sequences. Simultaneously, the mouse IgG subclass was determined at the DNA level based on isotype-specific sequence patterns in the CH1 domain. Knowing the DNA sequence of V-(D-)J recombination responsible for the complementary determining region 3 (CDR 3), variable region-specific primers were designed and used to amplify the corresponding antibody constant regions. Results: To verify the approach, we applied it to the hybridoma clone BAM-CCMV-29-81 and obtained identical full-length antibody sequences as with RNA Illumina sequencing. Further validation at the protein level using an established MALDI-TOF MS-fingerprinting protocol showed that five out of six genetically encoded CDR domains of the monoclonal antibody BAM-CCMV-29-81 could be efficiently correlated. Conclusion: This simple, streamlined method enables the cost-effective determination of the full-length sequence of monoclonal antibodies from hybridoma cell lines, with the added benefit of obtaining the DNA sequence of the antibody ready for recombinant expression. KW - Full-length antibody sequencing KW - Hybridoma cell loss KW - MALDI-TOF MS KW - Immunoglobulin isotyping KW - RNA Illumina sequencing PY - 2025 UR - https://nbn-resolving.org/urn:nbn:de:kobv:b43-639599 DO - https://doi.org/10.3390/antib14030072 SN - 2073-4468 VL - 14 IS - 3 SP - 1 EP - 17 PB - MDPI CY - Basel AN - OPUS4-63959 LA - eng AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER - TY - CONF A1 - Weller, Michael G. T1 - Antibody quality - a never-ending story? N2 - Deficiencies in research antibody quality lead to economic losses up to several billions of Euros annually. Poorly documented antibody experiments lead to a flood of publications without any chance to be reproduced and without any scientific value.The dominance of antibody specifications in complex analytical procedures is often highly underestimated. Most journals and funding agencies lack the expertise to identify major flaws in antibody experiments and their documentation. Most companies sell insufficiently characterized and documented antibodies. Recombinant antibodies are not the solution for antibody validation. T2 - Seminar des Instituts für Analytische Chemie der Universität Wien CY - Wien, Austria DA - 17.11.2016 KW - Antibodies KW - Reproducibility KW - Crisis KW - Antibody ID PY - 2016 AN - OPUS4-38424 LA - eng AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER - TY - JOUR A1 - Sauter, O. A1 - Neumann, Patrick P. A1 - Nerger, Tino A1 - Weller, Michael G. ED - Heß, M. T1 - Aus sicherer Entfernung BT - das Magazin für die Gefahrgut-Logistik N2 - Da chemische Gefahrstoffe im Boden eine ernsthafte Bedrohung für Leben und Gesundheit darstellen, das Grundwasser kontaminieren und langfristige Umweltschäden verursachen können, ist ihre frühzeitige Erkennung von großer Bedeutung. Um solche Gefahrstoffe aus sicherer Entfernung detektieren zu können, wurde bei der Bundesanstalt für Materialforschung und -prüfung (BAM) im Rahmen eines Forschungsvorhabens seit Anfang 2022 an einem neuen Ansatz zur Erkennung und Überwachung chemischer Gefahrstoffe gearbeitet. KW - Cellulose KW - Drohne KW - Kolorimetrische Sensoren KW - Passive Smart Dust PY - 2025 SN - 0016-5808 VL - 2025 IS - 8 SP - 26 EP - 28 PB - ecomed-Storck GmbH, Storck Verlag Hamburg CY - Hamburg AN - OPUS4-64109 LA - deu AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER - TY - CONF A1 - Tannenberg, Robert A1 - Paul, Martin A1 - Fünning, Tabea A1 - Schuhmann, Christoph A1 - Weller, Michael G. A1 - Steglich, Patrick T1 - Multichannel real-time detection of biomarkers with highly miniaturized photonic microchips N2 - The development of novel photonic integrated microchips (PIC) is a promising approach to allow for the convenient detection of key biomarkers in complex matrices through multichannel real-time analysis in a highly compact package. This study reports the successful development and application of a backside released CMOS chip designed for the multichannel real-time detection of biomarkers. Operating at the C-band at approx. 1550 nm, the microchip features three dedicated detection sensors in addition to a reference sensor, enabling simultaneous analysis of multiple biomarkers. The compact and highly miniaturized design of this microchip, with a footprint of just 1 mm², positions it as promising candidate for point-of-care diagnostics and personalized medicine applications. This technology opens a path to transform biomarker detection across various medical fields, offering rapid, reliable, and cost-effective diagnostic solutions. In conclusion, the presented multichannel photonic microchips signify a substantial leap forward in real-time biomarker detection, providing a highly capable platform for future research and clinical applications. T2 - SPIE Optics + Optoelectronics 2025 CY - Prague, Czech Republic DA - 07.04.2025 KW - Photonic integrated circuit KW - Ring resonator KW - Real-time detection KW - Multiplexing KW - Semiconductor KW - CMOS KW - C-reactive protein KW - CRP KW - Biomarker PY - 2025 SN - 978-1-5106-8850-6 DO - https://doi.org/10.1117/12.3056453 VL - 13527 SP - 1 EP - 6 PB - SPIE CY - Prague, Czech Republic AN - OPUS4-63478 LA - eng AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER - TY - JOUR A1 - Kaufmann, Jan Ole A1 - Brangsch, J. A1 - Kader, A. A1 - Saatz, Jessica A1 - Mangarova, D. B. A1 - Zacharias, M. A1 - Kempf, W. E. A1 - Schwaar, T. A1 - Ponader, Marco A1 - Adams, L. C. A1 - Möckel, J. A1 - Botnar, R. M. A1 - Taupitz, M. A1 - Mägdefessel, L. A1 - Traub, Heike A1 - Hamm, B. A1 - Weller, Michael G. A1 - Makowski, M. R. T1 - ADAMTS4-specific MR-probe to assess aortic aneurysms in vivo using synthetic peptide libraries N2 - The incidence of abdominal aortic aneurysms (AAAs) has substantially increased during the last 20 years and their rupture remains the third most common cause of sudden death in the cardiovascular field after myocardial infarction and stroke. The only established clinical parameter to assess AAAs is based on the aneurysm size. Novel biomarkers are needed to improve the assessment of the risk of rupture. ADAMTS4 (A Disintegrin And Metalloproteinase with ThromboSpondin motifs 4) is a strongly upregulated proteoglycan cleaving enzyme in the unstable course of AAAs. In the screening of a one-bead-one-compound library against ADAMTS4, a low-molecular-weight cyclic peptide is discovered with favorable properties for in vivo molecular magnetic resonance imaging applications. After identification and characterization, it’s potential is evaluated in an AAA mouse model. The ADAMTS4-specific probe enables the in vivo imaging-based prediction of aneurysm expansion and rupture. KW - Peptide KW - Peptide library KW - OBOC library KW - Combinatorial chemistry KW - Peptide aptamers KW - Binding molecule KW - Affinity KW - Synthetic peptides KW - Contrast agent KW - Magnetic resonance imaging KW - One-bead-one-compound library KW - On-chip screening KW - Lab-on-a-chip KW - MALDI-TOF MS KW - SPR KW - Surface plasmon resonance KW - Alanine scan KW - Fluorescence label KW - MST KW - Docking KW - Chelate PY - 2022 UR - https://nbn-resolving.org/urn:nbn:de:kobv:b43-560930 DO - https://doi.org/10.1038/s41467-022-30464-8 N1 - Geburtsname von Ponader, Marco: Wilke, M. - Birth name of Ponader, Marco: Wilke, M. VL - 13 IS - 1 SP - 1 EP - 18 PB - Springer Nature Limited CY - Heidelberg AN - OPUS4-56093 LA - eng AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER - TY - JOUR A1 - Weller, Michael G. T1 - A unifying review of bioassay-guided fractionation, effect-directed analysis and related techniques N2 - The success of modern methods in analytical chemistry sometimes obscures the problem that the ever increasing amount of analytical data does not necessarily give more insight of practical relevance. As alternative approaches, toxicity- and bioactivity-based assays can deliver valuable information about biological effects of complex materials in humans, other species or even ecosystems. However, the observed effects often cannot be clearly assigned to specific chemical compounds. In these cases, the establishment of an unambiguous cause-effect relationship is not possible. Effect-directed analysis tries to interconnect instrumental analytical techniques with a biological/biochemical entity, which identifies or isolates substances of biological relevance. Successful application has been demonstrated in many fields, either as proof-of-principle studies or even for complex samples. This review discusses the different approaches, advantages and limitations and finally shows some practical examples. The broad emergence of effect-directed analytical concepts might lead to a true paradigm shift in analytical chemistry, away from ever growing lists of chemical compounds. The connection of biological effects with the identification and quantification of molecular entities leads to relevant answers to many real life questions. KW - Toxicity KW - Drug discovery KW - Natural products KW - Environmental analysis KW - Lead identification PY - 2012 UR - https://nbn-resolving.org/urn:nbn:de:kobv:b43-262960 DO - https://doi.org/10.3390/s120709181 SN - 1424-8220 VL - 12 IS - 7 SP - 9181 EP - 9209 PB - MDPI CY - Basel AN - OPUS4-26296 LA - eng AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER - TY - JOUR A1 - Weller, Michael G. T1 - Immunoassays and biosensors for the detection of cyanobacterial toxins in water N2 - Algal blooms are a frequent phenomenon in nearly all kinds of fresh water. Global warming and eutrophication by waste water, air pollution and fertilizers seem to lead to an increased frequency of occurrence. Many cyanobacteria produce hazardous and quite persistent toxins, which can contaminate the respective water bodies. This may limit the use of the raw water for many purposes. The purification of the contaminated water might be quite costly, which makes a continuous and large scale treatment economically unfeasible in many cases. Due to the obvious risks of algal toxins, an online or mobile detection method would be highly desirable. Several biosensor systems have been presented in the literature for this purpose. In this review, their mode of operation, performance and general suitability for the intended purpose will be described and critically discussed. Finally, an outlook on current developments and future prospects will be given. KW - Algal toxins KW - Cyanotoxins KW - Biotoxins KW - Blue-green algae KW - Harmful algal blooms KW - Microcystins KW - Nodularins KW - Anatoxin-a KW - Anatoxin-a(s) KW - Cylindrospermopsin KW - Saxitoxins PY - 2013 UR - https://nbn-resolving.org/urn:nbn:de:kobv:b43-294728 DO - https://doi.org/10.3390/s131115085 SN - 1424-8220 VL - 13 IS - 11 SP - 15085 EP - 15112 PB - MDPI CY - Basel AN - OPUS4-29472 LA - eng AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER - TY - JOUR A1 - Hesse, Almut A1 - Weller, Michael G. T1 - Protein Quantification by Derivatization-Free High-Performance Liquid Chromatography of Aromatic Amino Acids N2 - Amino acid analysis is considered to be the gold standard for quantitative peptide and protein analysis. Here, we would like to propose a simple HPLC/UV method based on a reversed-phase separation of the aromatic amino acids tyrosine (Tyr), phenylalanine (Phe), and optionally tryptophan (Trp) without any derivatization. The hydrolysis of the proteins and peptides was performed by an accelerated microwave technique, which needs only 30 minutes. Two internal standard compounds, homotyrosine (HTyr) and 4-fluorophenylalanine (FPhe) were used for calibration. The limit of detection (LOD) was estimated to be 0.05 µM (~10 µg/L) for tyrosine and phenylalanine at 215 nm. The LOD for a protein determination was calculated to be below 16 mg/L (~300 ng BSA absolute). Aromatic amino acid analysis (AAAA) offers excellent accuracy and a precision of about 5% relative standard deviation, including the hydrolysis step. The method was validated with certified reference materials (CRM) of amino acids and of a pure protein (bovine serum albumin, BSA). AAAA can be used for the quantification of aromatic amino acids, isolated peptides or proteins, complex peptide or protein samples, such as serum or milk powder, and peptides or proteins immobilized on solid supports. KW - Protein analysis KW - Proteomics KW - Peptides KW - Metrology KW - Certified reference materials KW - Amino acid analysis KW - Hydrolysis KW - Microwave KW - Tyrosine KW - Phenylalanine KW - Tryptophan PY - 2016 UR - https://nbn-resolving.org/urn:nbn:de:kobv:b43-370215 DO - https://doi.org/10.1155/2016/7374316 SN - 2090-0112 SN - 2090-0104 VL - 2016 SP - Article 7374316, 1 EP - 8 PB - Hindawi CY - Cairo, London, New York AN - OPUS4-37021 LA - eng AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER - TY - JOUR A1 - Walter, Astrid A1 - Panne, Ulrich A1 - Weller, Michael G. T1 - A novel immunoreagent for the specific and sensitive detection of the explosive triacetone triperoxide (TATP) N2 - Triacetone triperoxide (TATP) is a primary explosive, which was used in various terrorist attacks in the past. For the development of biosensors, immunochemical µ-TAS, electronic noses, immunological test kits, or test strips, the availability of antibodies of high quality is crucial. Recently, we presented the successful immunization of mice, based on the design, synthesis, and conjugation of a novel TATP derivative. Here, the long-term immunization of rabbits is shown, which resulted in antibodies of extreme selectivity and more than 1,000 times better affinity in relation to the antibodies from mice. Detection limits below 10 ng L-1 (water) were achieved. The working range covers more than four decades, calculated from a precision profile. The cross-reactivity tests revealed an extraordinary selectivity of the antibodies—not a single compound could be identified as a relevant cross-reactant. The presented immunoreagent might be a major step for the development of highly sensitive and selective TATP detectors particularly for security applications. KW - Organic peroxides KW - Terrorism KW - Biosensor development PY - 2011 UR - https://nbn-resolving.org/urn:nbn:de:kobv:b43-240291 UR - http://www.mdpi.com/2079-6374/1/3/93 DO - https://doi.org/10.3390/bios1030093 SN - 2079-6374 VL - 1 IS - 3 SP - 93 EP - 106 PB - MDPI CY - Basel AN - OPUS4-24029 LA - eng AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER - TY - INPR A1 - Tchipilov, Teodor A1 - Meyer, Klas A1 - Weller, Michael G. T1 - Quantitative 1H nuclear magnetic resonance (qNMR) of aromatic amino acids for protein quantification 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 - JOUR A1 - Cervino, C. A1 - Knopp, D. A1 - Weller, Michael G. A1 - Niessner, R. T1 - Novel Aflatoxin derivatives and protein conjugates N2 - Aflatoxins, a group of structurally related mycotoxins, are well known for their toxic and carcinogenic effects in humans and animals. Aflatoxin derivatives and protein conjugates are needed for diverse analytical applications. This work describes a reliable and fast synthesis of novel aflatoxin derivatives, purification by preparative HPLC and characterisation by ESI-MS and one- and two-dimensional NMR. Novel aflatoxin bovine serum albumin conjugates were prepared and characterised by UV absorption and MALDI-MS. These aflatoxin protein conjugates are potentially interesting as immunogens for the generation of aflatoxin selective antibodies with novel specificities. KW - Aflatoxin KW - Mycotoxin KW - Hapten KW - Protein conjugation KW - Immunogen KW - Antigen KW - Antibody PY - 2007 UR - https://nbn-resolving.org/urn:nbn:de:kobv:b43-147572 UR - http://www.mdpi.org/molecules/papers/12030641.pdf UR - http://www.mdpi.org/molecules/papers/12030641sm.pdf DO - https://doi.org/10.3390/12030641 SN - 1420-3049 VL - 12 IS - 3 SP - 641 EP - 653 PB - MDPI CY - Basel AN - OPUS4-14757 LA - eng AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER - TY - JOUR A1 - Klykov, Oleg A1 - Weller, Michael G. T1 - Quantification of N-hydroxysuccinimide and N-hydroxysulfosuccinimide by hydrophilic interaction chromatography (HILIC) N2 - N-Hydroxysuccinimide (NHS) esters are the most important activated esters used in many different bioconjugation techniques, such as protein labelling by fluorescent dyes and enzymes, surface activation of chromatographic supports, microbeads, nanoparticles, and microarray slides, and also in the chemical synthesis of peptides. Usually, reactions with NHS esters are very reliable and of high yield, however, the compounds are sensitive to air moisture and water traces in solvents. Therefore, the quantification of NHS would be a very helpful approach to identify reagent impurities or degradation of stored NHS esters. No robust and sensitive method for the detection of NHS (or the more hydrophilic sulfo-NHS) has been reported yet. Here, a chromatographic method based on HILIC conditions and UV detection is presented, reaching a detection limit of about 1 mg L-1, which should be sensitive enough for most of the applications mentioned above. Exemplarily, the hydrolytic degradation of a biotin-NHS ester and a purity check of a fluorescent dye NHS ester are shown. An important advantage of this approach is its universality, since not the structurally variable ester compound is monitored, but the constant degradation product NHS or sulfo-NHS, which avoids the necessity to optimize the separation conditions and facilitates calibration considerably. KW - NHS KW - Sulfo-NHS KW - NHS-Ester KW - Qualitätskontrolle KW - Reinheitsbestimmung KW - HPLC PY - 2015 UR - https://nbn-resolving.org/urn:nbn:de:kobv:b43-337833 DO - https://doi.org/10.1039/c5ay00042d SN - 1759-9660 SN - 1759-9679 VL - 7 IS - 15 SP - 6443 EP - 6448 PB - RSC Publ. CY - Cambridge AN - OPUS4-33783 LA - eng AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER - TY - CONF A1 - Weller, Michael G. T1 - Antibody-based SAW sensor for the detection of explosives N2 - A robust and sensitive method for the detection of the explosive trinitrotoluene (TNT) was developed. The detection limit was determined to be around 0.5 µg/L. The fast signal response of less than 1 minute shows that this approach is suitable for security and other time-critcal applications. In addition, the very low cross-reactivity highly reduces the number of false-positives in relation to competing techniques, including sniffer dogs. Due to the multianalyte ability of the SAW system, several explosives might be detected in parallel. T2 - BAM Meeting 2016 CY - Berlin, Germany DA - 17.02.2016 KW - TNT KW - Trinitrotoluene KW - Explosives KW - Airport security KW - High-speed biosensor KW - Selectivity KW - Sensitivity KW - Self-assembled monolayer KW - Gold surface KW - Immunosensor KW - Reversibility KW - Inhibition assay KW - Polyethylene glycol KW - PEG PY - 2016 UR - https://nbn-resolving.org/urn:nbn:de:kobv:b43-366265 UR - http://f1000research.com/posters/5-1352 DO - https://doi.org/10.7490/f1000research.1112309.1 AN - OPUS4-36626 LA - eng AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER - TY - CONF A1 - Weller, Michael G. T1 - Identification of toxicants in complex environmental samples using HPLC combined online with toxicity testing T2 - Anakon 2007 CY - Jena, Germany DA - 2007-03-27 PY - 2007 AN - OPUS4-14737 LA - eng AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER - TY - JOUR A1 - Giesen, Charlotte A1 - Jakubowski, Norbert A1 - Panne, Ulrich A1 - Weller, Michael G. T1 - Comparison of ICP-MS and photometric detection of an immunoassay for the determination of ochratoxin A in wine N2 - An immunoassay was developed for ochratoxin A (OTA) detection in wine and two completely different detection methods were compared: ICP-MS and photometry. For labelling, we applied secondary antibodies conjugated with gold nanoparticles for ICP-MS and with horseradish peroxidase for photometric detection, respectively. Detection limits of the assay were determined as 0.003 µg L-1 for both detection methods. OTA in wine was determined below the EU limit value of 2 µg kg-1. The measurement range was between 0.01 and 1 µg L-1 for ICP-MS and photometric detection. Assay precision detected by ICP-MS and photometry were similar showing that precision of the detection method has only a minor effect on total assay precision. By using BSA as a buffer additive to minimize nonspecific binding, we could also confirm that OTA strongly binds to BSA. In a comparison study of several additives, casein was found to be preferable. PY - 2010 DO - https://doi.org/10.1039/c0ja00009d SN - 0267-9477 SN - 1364-5544 VL - 25 IS - 10 SP - 1567 EP - 1572 PB - Royal Society of Chemistry CY - London AN - OPUS4-22704 LA - eng AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER - TY - JOUR A1 - Pecher, Heike Susanne A1 - Zimathies, Annett A1 - Weller, Michael G. T1 - Oligoepoxide-based monoliths: synthesis and application as affinity capillary column for enrichment of immunoglobulin G N2 - A series of new epoxide-based monoliths is synthesized by self-polymerization of polyglycerol polyglycidyl ether as porous support for affinity chromatography. Porosity and mean pore sizes are investigated as a function of the porogen ratio and analyzed by mercury porosimetry. The morphologies are visualized by environmental scanning electron microscope. With a particularly rigid monolith prepared with 75 vol% porogen (66% porosity and 12 µm mean pore size), a capillary column (0.5 mm ID) is prepared and directly functionalized with recombinant protein A to extract immunoglobulin G from rabbit serum. KW - Affinity chromatography KW - Affinity extraction KW - Glycidylethers KW - Macroporous polymers KW - Polyethers PY - 2012 DO - https://doi.org/10.1002/macp.201200206 SN - 1022-1352 SN - 1521-3935 VL - 213 IS - 22 SP - 2398 EP - 2403 PB - Wiley-VCH Verl. CY - Weinheim AN - OPUS4-27390 LA - eng AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER - TY - JOUR A1 - Carvalho, José Joao A1 - Weller, Michael G. A1 - Panne, Ulrich A1 - Schneider, Rudolf T1 - Monitoring caffeine in human saliva using a newly developed ELISA N2 - Caffeine is a useful indicator to quickly assess liver function. High-throughput tests are needed for single-point caffeine measurements, with low cross-reactivity toward its major metabolite, paraxanthine. A newly developed ELISA was compared with an LC-MS/MS reference method, using 60 saliva samples from 10 individuals, before and after caffeine intake. Bland-Altman plot, Student t-test and F-test were used to compare the two methods. Proteins were precipitated using organic solvent and the caffeine recoveries compared with those obtained using sample microfiltration. The antibody, with a low cross-reactivity toward paraxanthine (0.08%), allows quantification of caffeine in saliva samples from 2.5 µg/L to 125 µg/L with high precision and the ELISA shows comparable results to those obtained by LC-MS/MS. A one-step protein precipitation using an organic solvent provides comparable results to a more costly and time-consuming microfiltration pre-treatment of samples. The new ELISA is a fit-for-purpose method to accurately and precisely determine caffeine in saliva samples. KW - Caffeine KW - Cytochrome P450 CYP1A2 KW - Enzyme-linked immunosorbent assay KW - LC-MS/MS KW - Liver function tests KW - Saliva PY - 2012 DO - https://doi.org/10.1080/00032719.2012.696226 SN - 0003-2719 SN - 1532-236X VL - 45 IS - 17 SP - 2549 EP - 2561 PB - Taylor & Francis CY - Philadelphia, Pa. AN - OPUS4-27267 LA - eng AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER -