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Eingeladener Vortrag
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Im Vorhaben „MARKERIA - Bestimmung von anthropogenen Markerkonzentrationen für die SARS-CoV-2 Quantifizierung mittels eines Hochdurchsatzverfahrens (ELISA)“ geht es um die Bestimmung von Carbamazepin, Diclofenac, Koffein und einer endogenen Gallensäure als potentielle Marker für die Kalibrierung der SARS-CoV-2 Quantifizierung im Abwasser. Es ist ein Teilvorhaben des übergeordneten Projektes ESI-CorA und wird von der BAM im Auftrag des Umweltbundesamtes durchgeführt. Der Vortrag wurde gehalten im Rahmen der Abschlussveranstaltung des Projektes ESI-CorA. Er gab den Projektstand wieder, den UBA und BAM bis dahin erzielt hatten, das SARS-CoV-2 Virus und die anthropogenen Marker in 20 Kläranlagenzuläufen zu bestimmen und miteinander zu korrelieren.
Im Vorhaben „MARKERIA - Bestimmung von anthropogenen Markerkonzentrationen für die SARS-CoV-2 Quantifizierung mittels eines Hochdurchsatzverfahrens (ELISA)“ geht es um die Bestimmung von Carbamazepin, Diclofenac, Koffein und einer endogenen Gallensäure als potentielle Marker für die Kalibrierung der SARS-CoV-2 Quantifizierung im Abwasser. Der Vortrag wurde gehalten im Rahmen der 22. Sitzung des Begleitkreises des übergeordneten Projektes ESI-CorA. Er gab den Projektstand wieder, den die BAM bis dahin erzielt hatte. Die Marker waren in sämtlichen untersuchten Abwasser-Zulaufproben enthalten und zeigten spezifische Trends. Weitere Korrelationen müsse die weitergehende Datenauswertung ergeben.A
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.
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.
In coastal systems, organisms are exposed to amultitude of stressors whose interactions and effects are poorly studied. Pharmaceutical drugs and Climate Change consequences, such as lowered pH, are examples of stressors affecting marine organisms, as bivalves. Although a vast literature is available for the effects of these stressors when acting individually, very limited information exists on the impacts that the combination of both can have on marine bivalves. For this reason, this study aimed to evaluate the impacts of a simulated ocean acidification scenario (control pH, 8.0; lowered pH, pH 7.6) on the effects of the antiepileptic carbamazepine (CBZ, 1 μg/L) and the antihistamine cetirizine (CTZ, 0.6 μg/L), when acting individually and combined (CBZ + CTZ), on the edible clam Ruditapes philippinarum. After 28 days of exposure, drug concentrations, bioconcentration factors and biochemical parameters related to the clams' metabolic capacity and oxidative stress were evaluated. The results showed that R. philippinarum clams responded differently to pharmaceutical drugs depending on the pH tested, influencing both bioconcentration and biological responses. In general, drug combined treatments showed fewer impacts than drugs acting alone, and acidification seemed to activate at a higher extension the elimination processes that were not activated under control pH. Also, lowered pH per se exerted negative impacts (e.g., cellular damage) on R. philippinarum and the combination with pharmaceutical drugs did not enhance the toxicity.
In Coastal Systems, pollutants as pharmaceutical drugs exert changes from the molecular to the organism level in marine bivalves. Besides pollutants, Coastal Systems are prone to changes in environmental Parameters, as the alteration of salinity values because of Climate Change. Together, these Stressors (pharmaceutical drugs and salinity changes) can exert different threats than each Stressor acting individually; for example, salinity can change the physical-chemical properties of the drugs and/or the sensitivity of the organisms to them. However, limited Information is available on this subject, with variable results, and for this reason, this study aimed to evaluate the impacts of salinity changes (15,25 and 35) on the effects of the antiepileptic carbamazepine (CBZ, 1 (ig/L) and the antihistamine cetirizine (CTZ, 0.6 pg/L), when acting individually and combined (CBZ + CTZ), in the edible clam Ruditapes philippinarum. After 28 days ofexposure, drugs concentrations, bioconcentration factors and biochemical parameters, related to clam's metabolic caparity and oxidative stress were evaluated. The results showed that dams under low salinity suffered more changes in metabolic, antioxidant and biotransformation activities, in comparison with the remaining salinities under study. However, limited impacts were observed when comparing drug effects at low salinity. Indeed, it seemed that CTZ and CBZ + CTZ, under high salinity (salinity 35) were the worst exposure conditions for the dams, since they caused higher leveis of cellular damage. It Stands out that salinity changes altered the impact of pharmaceutical drugs on marine bivalves.
Vergleichende Darstellung SARS-CoV-2-spezifischer Nanobodys aus unterschiedlichen Wirtsorganismen
(2021)
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.
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.
A vast number of emerging pollutants is being detected in the environment. Another lingering problem are health-threatening contaminants, such as mycotoxins, that deteriorate food and feed.
Analytical methods, suitable for trace analysis, are needed that are desirably also fast, inexpensive and, if possible, robust, and portable. Immunoanalytical, i.e., anti-body-based technologies, which are available in a broad range of formats, can be profitably used here to analyze for the distribution and the concentration trends of contaminants.
Some of these formats are single-analyte but high-throughput methods. To use them wisely, indicator substances or sum parameters must be established to be used in screening approaches. Other methods are suitable to be performed on portable in-strumentation in the field (on-site), more precisely at the point-of-need. Furthermore, there are array technologies that allow for parallel analysis of several analytes of interest (multiplexing). The talk reports on methods and data for compounds con-sidered as anthropogenic contamination markers (the antiepileptic carbamazepine, the analgesic diclofenac, the anti-histaminic cetirizine, the steroid hormone estrone, the antimicrobial amoxicillin, the stimulants caffeine and cocaine, the endocrine disruptor bisphenol A, and a bile acid). Some work on mycotoxins (ochratoxin A and ergot alkaloids) is also presented. It is demonstrated that antibody-based technolo-gies have a huge potential to gain analytical insights at the point-of-need.