TY - JOUR A1 - Walter, Astrid A1 - Pfeifer, Dietmar A1 - Kraus, Werner A1 - Emmerling, Franziska A1 - Schneider, Rudolf A1 - Panne, Ulrich A1 - Weller, Michael G. T1 - Triacetone Triperoxide (TATP): Hapten design and development of antibodies N2 - Triacetone triperoxide (TATP), an improvised explosive, is a potential security threat because of its cost-efficient synthesis and the difficulty in detecting it. A highly selective antibody could provide the necessary specificity to the detection process. To obtain antibodies, a hapten made from acetone, hydrogen peroxide, and 7-oxooctanoic acid has been designed, synthesized, and confirmed by NMR that displays the utmost similarity to the analyte. The single-crystal X-ray structures of the solvated species TATP·methanol (1:1) and the TATP derivate were determined. In both compounds, the molecules exhibit D3 symmetry and adopt a twisted boat-chair conformation. The hapten was coupled to bovine serum albumin, and mice were immunized. An immune response against TATP was elicited, and selective antibodies were detected in the mouse serum, which should be very useful for the development of a TATP biosensor system. An ELISA with a limit of detection for TATP of 65 µg L-1 is shown. KW - TATP KW - Triacetone triperoxide KW - Explosive KW - Antibody KW - Hapten immunoassay KW - Structure PY - 2010 DO - https://doi.org/10.1021/la1018339 SN - 0743-7463 SN - 1520-5827 VL - 26 IS - 19 SP - 15418 EP - 15423 PB - American Chemical Society CY - Washington, DC AN - OPUS4-22469 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 - Carvalho, José Joao A1 - Walter, Astrid A1 - Baermann, Yvonne A1 - Weller, Michael G. A1 - Panne, Ulrich A1 - Schenk, J.A. A1 - Schneider, Rudolf T1 - Non-invasive monitoring of immunization progress in mice via IgG from feces N2 - A non-invasive method to monitor the humoral immune response in mice after immunization is described. From fecal pellets of an individual mouse, a sufficient amount of active immunoglobulins or their fragments can be extracted to perform a regular examination of the status of the immune response by immunoassay. Hapten-specific antibodies from the feces of mice from three immunization trials showed very similar characteristics to those obtained from serum at a given date. Therefore, it can be suspected that some serum IgG enters the intestinal lumen and ends up in the feces, where they appear to be considerably stable. Hapten-specific IgAs were not found in the feces. Being able to analyze antibody titers in feces could be an interesting animal welfare refinement to standard practice that does not entail repeated blood sampling. KW - Immunization monitoring KW - IgG KW - Coproantibodies KW - Feces KW - Animal welfare KW - 3R concept PY - 2012 UR - http://iv.iiarjournals.org/content/26/1/63.full.pdf+html SN - 0258-851x VL - 26 IS - 1 SP - 63 EP - 70 PB - In vivo CY - Athens AN - OPUS4-25283 LA - eng AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER - TY - JOUR A1 - Bahlmann, Arnold A1 - Weller, Michael G. A1 - Panne, Ulrich A1 - Schneider, Rudolf T1 - Monitoring carbamazepine in surface and wastewaters by an immunoassay based on a monoclonal antibody N2 - The pharmaceutical compound carbamazepine (CBZ) is an emerging pollutant in the aquatic environment and may potentially be used as a wastewater marker. In this work, an enzyme-linked immunosorbent assay (ELISA) for the detection of carbamazepine in surface and sewage waters has been developed. The heterogeneous immunoassay is based on a commercially available monoclonal antibody and a novel enzyme conjugate (tracer) that links the hapten via a hydrophilic peptide (triglycine) spacer to horseradish peroxidase. The assay achieves a limit of detection of 24 ng/L and a quantitation range of 0.05-50 µg/L. The analytical performance and figure of merits were compared to liquid chromatography-tandem mass spectrometry after solid-phase extraction. For nine Berlin surface water samples and one wastewater sample, a close correlation of results was observed. A constant overestimation relative to the CBZ concentration of approximately 30% by ELISA is probably caused by the presence of 10,11-epoxy-CBZ and 2-hydroxy-CBZ in the samples. The ELISA displayed cross-reactivities for these compounds of 83% and 14%, respectively. In a first screening of 27 surface water samples, CBZ was detected in every sample with concentrations between 0.05 and 3.2 µg/L. Since no sample cleanup is required, the assay allowed for the determination of carbamazepine with high sensitivity at low costs and with much higher throughput than with conventional methods. KW - Carbamazepine KW - ELISA KW - Antibody KW - Immunoassay KW - Surface water KW - LC-MS/MS KW - Pharmaceuticals PY - 2009 DO - https://doi.org/10.1007/s00216-009-2958-7 SN - 1618-2642 SN - 1618-2650 VL - 395 IS - 6 SP - 1809 EP - 1820 PB - Springer CY - Berlin AN - OPUS4-20927 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 - TY - JOUR A1 - Bahlmann, Arnold A1 - Carvalho, José Joao A1 - Weller, Michael G. A1 - Panne, Ulrich A1 - Schneider, Rudolf T1 - Immunoassays as high-throughput tools: monitoring spatial and temporal variations of carbamazepine, caffeine and cetirizine in surface and wastewaters N2 - Carbamazepine (CBZ), caffeine and cetirizine were monitored by enzyme-linked immunosorbent assays (ELISAs) in surface and wastewaters from Berlin, Germany. This fast and cost-efficient method enabled to assess the spatial and temporal variation of these anthropogenic markers in a high-throughput screening. CBZ and cetirizine were detected by the same antibody, which selectively discriminates between both compounds depending on the pH value used in the incubation step. To our best knowledge, this is the first dual-analyte immunoassay working with a single antibody. The frequent sampling with 487 samples being processed allowed for the repeated detection of unusually high concentrations of CBZ and caffeine. ELISA results correlate well with the ones obtained by liquid chromatography tandem mass spectrometry (LC-MS/MS). Caffeine concentrations found in surface waters were elevated by combined sewer overflows after stormwater events. During the hay fever season, the concentrations of the antihistamine drug cetirizine increased in both surface and wastewaters. Caffeine was almost completely removed during wastewater treatment, while CBZ and cetirizine were found to be more persistent. The maximum concentrations of caffeine, CBZ and cetirizine found in influent wastewater by LC–MS/MS were 470, 5.0 and 0.49 µg L-1, while in effluent wastewater the concentrations were 0.22, 4.5 and 0.51 µg L-1, respectively. For surface waters, concentrations up to 3.3, 4.5 and 0.72 µg L-1 were found, respectively. KW - ELISA KW - Carbamazepine KW - Caffeine KW - Cetirizine KW - Environmental monitoring KW - Anthropogenic markers PY - 2012 DO - https://doi.org/10.1016/j.chemosphere.2012.05.020 SN - 0045-6535 SN - 0366-7111 VL - 89 IS - 11 SP - 1278 EP - 1286 PB - Elsevier Science CY - Kidlington, Oxford AN - OPUS4-26187 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 - A highly sensitive caffeine immunoassay based on a monoclonal antibody KW - Caffeine KW - ELISA KW - Antibody KW - LC–MS–MS KW - Water KW - Wastewater marker PY - 2010 DO - https://doi.org/10.1007/s00216-010-3506-1 SN - 1618-2642 SN - 1618-2650 VL - 396 IS - 7 SP - 2617 EP - 2628 PB - Springer CY - Berlin AN - OPUS4-22651 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 - JOUR A1 - Bosc-Bierne, Gaby A1 - Ewald, Shireen A1 - Kreuzer, Oliver J. A1 - Weller, Michael G. T1 - Efficient Quality Control of Peptide Pools by UHPLC and Simultaneous UV and HRMS Detection N2 - Peptide pools consist of short amino acid sequences and have proven to be versatile tools in various research areas in immunology and clinical applications. They are commercially available in many different compositions and variants. However, unlike other reagents that consist of only one or a few compounds, peptide pools are highly complex products which makes their quality control a major challenge. Quantitative peptide analysis usually requires sophisticated methods, in most cases isotope-labeled standards and reference materials. Usually, this would be prohibitively laborious and expensive. Therefore, an approach is needed to provide a practical and feasible method for quality control of peptide pools. With insufficient quality control, the use of such products could lead to incorrect experimental results, worsening the well-known reproducibility crisis in the biomedical sciences. Here we propose the use of ultra-high performance liquid chromatography (UHPLC) with two detectors, a standard UV detector at 214 nm for quantitative analysis and a high-resolution mass spectrometer (HRMS) for identity confirmation. To be cost-efficient and fast, quantification and identification are performed in one chromatographic run. An optimized protocol is shown, and different peak integration methods are compared and discussed. This work was performed using a peptide pool known as CEF advanced, which consists of 32 peptides derived from cytomegalovirus (CMV), Epstein–Barr virus (EBV) and influenza virus, ranging from 8 to 12 amino acids in length. N2 - Peptidpools bestehen aus kurzen Aminosäuresequenzen und haben sich als vielseitige Werkzeuge in verschiedenen Forschungsbereichen der Immunologie und bei klinischen Anwendungen erwiesen. Sie sind in vielen verschiedenen Zusammensetzungen und Varianten im Handel erhältlich. Im Gegensatz zu anderen Reagenzien, die nur aus einer oder wenigen Verbindungen bestehen, sind Peptidpools jedoch hochkomplexe Produkte, was ihre Qualitätskontrolle zu einer großen Herausforderung macht. Die quantitative Peptidanalyse erfordert in der Regel ausgefeilte Methoden, in den meisten Fällen isotopenmarkierte Standards und Referenzmaterialien. Dies ist in der Regel sehr aufwändig und teuer. Daher wird ein Ansatz benötigt, der eine praktische und praktikable Methode zur Qualitätskontrolle von Peptidpools bietet. Bei unzureichender Qualitätskontrolle könnte die Verwendung solcher Produkte zu falschen Versuchsergebnissen führen, was das bekannte Problem der Reproduzierbarkeit in den biomedizinischen Wissenschaften noch verschärfen würde. Hier schlagen wir die Verwendung der Ultrahochleistungs-Flüssigkeitschromatographie (UHPLC) mit zwei Detektoren vor, einem Standard-UV-Detektor bei 214 nm für die quantitative Analyse und einem hochauflösenden Massenspektrometer (HRMS) für die Identitätsbestätigung. Um kosteneffizient und schnell zu sein, werden Quantifizierung und Identifizierung in einem einzigen chromatographischen Lauf durchgeführt. Es wird ein optimiertes Protokoll gezeigt, und es werden verschiedene Peak-Integrationsmethoden verglichen und diskutiert. Für diese Arbeit wurde ein Peptidpool verwendet, der als CEF advanced bekannt ist und aus 32 Peptiden besteht, die vom Cytomegalovirus (CMV), Epstein-Barr-Virus (EBV) und Influenzavirus stammen und zwischen 8 und 12 Aminosäuren lang sind. KW - Synthetic peptides KW - Quality control KW - Impurites KW - Byproducts KW - Degradation KW - Mass spectrometry KW - Orbitrap PY - 2024 UR - https://nbn-resolving.org/urn:nbn:de:kobv:b43-602299 DO - https://doi.org/10.3390/separations11050156 SN - 2297-8739 VL - 11 IS - 5 SP - 1 EP - 18 PB - MDPI CY - Basel AN - OPUS4-60229 LA - eng AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER - TY - JOUR A1 - Fischer, Janina A1 - Kaufmann, Jan Ole A1 - Weller, Michael G. T1 - Simple Determination of Affinity Constants of Antibodies by Competitive Immunoassays N2 - The affinity constant, also known as the equilibrium constant, binding constant, equilibrium association constant, or the reciprocal value, the equilibrium dissociation constant (Kd), can be considered as one of the most important characteristics for any antibody–antigen pair. Many methods based on different technologies have been proposed and used to determine this value. However, since a very large number of publications and commercial datasheets do not include this information, significant obstacles in performing such measurements seem to exist. In other cases where such data are reported, the results have often proved to be unreliable. This situation may indicate that most of the technologies available today require a high level of expertise and effort that does not seem to be available in many laboratories. In this paper, we present a simple approach based on standard immunoassay technology that is easy and quick to perform. It relies on the effect that the molar IC50 approaches the Kd value in the case of infinitely small concentrations of the reagents. A two-dimensional dilution of the reagents leads to an asymptotic convergence to Kd. The approach has some similarity to the well-known checkerboard titration used for the optimization of immunoassays. A well-known antibody against the FLAG peptide, clone M2, was used as a model system and the results were compared with other methods. This approach could be used in any case where a competitive assay is available or can be developed. The determination of an affinity constant should belong to the crucial parameters in any quality control of antibody-related products and assays and should be mandatory in papers using immunochemical protocols. N2 - Die Affinitätskonstante, auch bekannt als Gleichgewichtskonstante, Bindungskonstante, Gleichgewichtsassoziationskonstante oder der reziproke Wert, die Gleichgewichtsdissoziationskonstante (Kd), kann als eine der wichtigsten Eigenschaften für jedes Antikörper-Antigen-Paar angesehen werden. Zur Bestimmung dieses Wertes wurden zahlreiche Methoden auf der Grundlage verschiedener Technologien vorgeschlagen und verwendet. Da jedoch eine sehr große Anzahl von Veröffentlichungen und kommerziellen Datenblätter diese Information nicht enthalten, scheint es erhebliche Hindernisse bei der Durchführung solcher Messungen zu geben. In anderen Fällen, in denen solche Daten angegeben werden, haben sich die Ergebnisse häufig als unzuverlässig erwiesen. Diese Situation könnte darauf hindeuten, dass die meisten der heute verfügbaren Technologien ein hohes Maß an Fachwissen und Aufwand erfordern, das in vielen Labors nicht vorhanden zu sein scheint. In diesem Beitrag stellen wir einen einfachen Ansatz vor, der auf der Standard-Immunoassay-Technologie basiert und einfach und schnell durchführbar ist. Er beruht auf dem Effekt, dass sich der molare IC50 bei unendlich kleinen Reagenz-Konzentrationen dem Kd-Wert annähert. Eine zweidimensionale Verdünnung der Reagenzien führt zu einer asymptotischen Konvergenz zu Kd. Der Ansatz hat eine gewisse Ähnlichkeit mit der bekannten Checkerboard-Titration, die zur Optimierung von Immunoassays verwendet wird. Ein bekannter Antikörper gegen das FLAG-Peptid, Klon M2, wurde als Modellsystem verwendet, und die Ergebnisse wurden mit anderen Methoden verglichen. Dieser Ansatz kann überall dort angewendet werden, wo ein kompetitiver Assay verfügbar ist oder entwickelt werden kann. Die Bestimmung einer Affinitätskonstante sollte zu den entscheidenden Parametern bei der Qualitätskontrolle von Antikörperprodukten und -assays gehören und in Arbeiten, die immunchemische Protokolle verwenden, obligatorisch sein. KW - ELISA KW - Competitive immunoassay KW - IC50 KW - Test midpoint KW - Point of inflection KW - Equilibrium constant KW - Dissociation constant KW - Binding strength KW - Antibody antigen complex KW - Bio-interaction KW - SPR KW - Surface-plasmon resonance KW - FLAG peptide KW - Clone M2 KW - Antibody quality PY - 2024 UR - https://nbn-resolving.org/urn:nbn:de:kobv:b43-605607 DO - https://doi.org/10.3390/mps7030049 SN - 2409-9279 VL - 7 IS - 3 SP - 1 EP - 11 PB - MDPI CY - Basel AN - OPUS4-60560 LA - eng AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER -