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Under the auspices of the Protein Analysis Working Group (PAWG) of the Comité Consultatif pour la Quantité de Matière (CCQM) a pilot study, CCQM-P216, was coordinated by the Chinese National Institute of Metrology (NIM), National Research Council of Canada (NRC) and the Bureau International des Poids et Mesures (BIPM). Eleven Metrology Institutes or Designated Institutes and the BIPM participated in the first phase of the pilot study (Part 1). The purpose of this pilot study was to develop measurement capabilities for larger proteins using a recombinant humanized IgG monoclonal antibody against Spike glycoprotein of SARS-CoV-2 (Anti-S IgG mAb) in solution. The first phase of the study was designed to employ established methods that had been previously studies by the CCQM Protein Analysis Working Group, involving the digestion of protein down to the peptide or amino acid level. The global coronavirus pandemic has also led to increased focus on antibody quantitation methods. IgG are among the immunoglobulins produced by the immune system to provide protection against SARS-CoV-2. Anti-SARS-CoV-2 IgG can therefore be detected in samples from affected patients. Antibody tests can show whether a person has been exposed to the SARS-CoV-2, and whether or not they potentially show lasting immunity to the disease. With the constant spread of the virus and the high pressure of re-opening economies, antibody testing plays a critical role in the fight against COVID-19 by helping healthcare professionals to identify individuals who have developed an immune response, either via vaccination or exposure to the virus. Many countries have launched large-scale antibody testing for COVID-19. The development of measurement standards for the antibody detection of SARS-CoV-2 is critically important to deal with the challenges of the COVID-19 pandemic. In this study, the SARS-CoV-2 monoclonal antibody is being used as a model system to build capacity in methods that can be used in antibody quantification. Amino acid reference values with corresponding expanded uncertainty of 36.10 ± 1.55 mg/kg, 38.75 ± 1.45 mg/kg, 18.46 ± 0.78 mg/kg, 16.20 ± 0.67 mg/kg and 30.61 ± 1.30 mg/kg have been established for leucine, valine, phenylalanine, isoleucine and proline, respectively. Agreement between nearly all laboratories was achieved for the amino acid analysis within 2 to 2.5 %, with one participant achieving markedly higher results due to a technical issue found in their procedure; this result was thus excluded from the reference value calculations. The relatively good agreement within a laboratory between different amino acids was not dissimilar to previous results for peptides or small proteins, indicating that factors such as hydrolysis conditions and calibration procedures could be the largest sources of variability. Peptide reference values with corresponding expanded uncertainty of 4.99 ± 0.28 mg/kg and 6.83 ± 0.65 mg/kg have been established for ALPAPIEK and GPSVFPLAPSSK, respectively. Not surprisingly due to prior knowledge from previous studies on peptide quantitation, agreement between laboratories for the peptide-based analysis was slightly poorer at 3 to 5 %, with one laboratory's result excluded for the peptide GPSVFPLAPSSK. Again, this level of agreement was not significantly poorer than that achieved in previous studies with smaller or less complex proteins. To reach the main text of this paper, click on Final Report.
In this work, we report an electrochemical immunosensor to detect ethinylestradiol in water samples, using electrochemical impedance spectroscopy (EIS) as a detection technique. For the development of this immunosensor, the direct modification of the working electrode of a screen-printed carbon electrode was carried out. First, to reduce the resistance of the electrode, electroreduced graphene was incorporated on the surface.
Second, a porous gold structure was electrodeposited on reduced graphene by electrodeposition and the dynamic hydrogen bubble template assisted method. Thus, a marked increase in surface area was obtained for anti-EE2 antibodies immobilization. Subsequently, the specific anti-EE2 antibodies were covalently immobilized using α-lipoic acid for attaching them to the gold surface.
The electrode modified with the antibodies was incubated for 30 min in the samples containing EE2, producing the specific Antigen antibody binding. As the charge transfer resistance of a redox probe in the electrode surface is governed by the surface blocking effects, the charge transfer resistance was related to the amount of EE2 captured to realize a quantitative determination. For this, the EIS measurements were performed in a 4 mM [Fe(CN)6]4−/3− solution in 0.1 M KCl. The obtained Nyquist diagrams were adjusted using the Randles circuit as an equivalent circuit to obtain the corresponding resistances. The developed methodology showed good selectivity, precision, and sensitivity; although the LOD obtained was higher than those presented in other published articles, it turned out to be an alternative that allows the determination of ethinylestradiol using a simple disposable electrode.
In this work we report a novel paper-based analytical device read-out via LED-induced fluorescence detection (FPAD) for the quantification of the emerging pollutant ethinylestradiol (EE2) in river water samples. The PAD was used as a reaction platform for a competitive enzyme immunoassay. For the PAD development, microzones of filter paper, printed by a wax printing method, were modified with amino-functionalized SBA-15 and subsequently, anti-EE2 specific antibodies were covalently immobilized. The determination of EE2 in water was
carried out by adding a fixed concentration of EE2 conjugated with the enzyme horseradish peroxidase (HRP) to samples and standards. Then, the FPAD were added and incubated for 10 min. Finally, the detection was performed by the reaction of 10-acetyl-3,7-dihydroxyphenoxazine (ADHP) whose oxidation is catalyzed by HRP in the presence of H2O2, obtaining the highly fluorescent resorufin (R). Resorufin was detected by LED excitation at 550 nm, observing emission at 585 nm. The EE2 concentration in the samples was inversely proportional to the relative fluorescence obtained from the enzymatic reaction products. The FPAD assay showed a detection Limit (LOD) of 0.05 ng L−1 and coefficients of variation (CV) below 4.5% within-assay and below 6.5% between-assay, respectively. The results obtained show the potential suitability of our FPAD for the selective and sensitive quantification of EE2 in river water samples. In addition, it has the PADs advantages of being disposable, easy to
apply and inexpensive.
We report a novel and innovative electrochemical paper-based immunocapture assay (EPIA) to address the need for ultrasensitive detection of emerging pollutants without regulatory status and whose effects on environment and human health are not completely yet understood. In particular, we present the application of this system toward highly sensitive detection of the emerging pollutant ethinyl estradiol (EE2). The EPIA approach is based on the use of paper microzones modified with silica nanoparticles (SNs) and anti-EE2 specific antibodies for capture and preconcentration of EE2 from river water samples. After the preconcentration procedure, the paper microzones are placed onto a screen-printed carbon electrode modified with electrochemically reduced graphene (RG). The bound EE2 is subsequently desorbed adding a diluted solution of sulfuric acid on the paper microzones. Finally, recovered EE2 is electrochemically detected by OSWV. The proposed novel methodology showed an appropriate LOD and linear range for the quantification of EE2 for water samples with different origins. The nonsophisticated equipment required, the adequate recovery values obtained (from 97% to 104%, with a RSD less than 4.9%), and the appropriate LOD and linear range value (0.1 ng L−1 and 0.5−120 ng L−1, respectively) achieved by our immunocapture sensor present significant analytical figures of merit, particularly when the routine quantification of EE2 is considered. In addition, our System was based on electrochemical paper-based technology, which allows obtainment of portable, easy-to-use, inexpensive, and disposable devices. The EPIA can also serve as a general-purpose immunoassay platform applicable to quantitation of other drugs and emerging pollutants in environmental samples.
Here, we use cryo soft X-ray tomography (cryo-SXT), which delivers 3D ultrastructural volumes of intact cells without chemical fixation or staining, to gain insight about nanoparticle uptake for nanomedicine. We initially used dendritic polyglycerol sulfate (dPGS) with potential diagnostic and therapeutic applications in inflammation. Although dPGS-coated gold nanoparticle (dPGS-AuNP) uptake followed a conventional endocytic/degradative pathway in human lung epithelial cell lines (A549), with cryo-SXT, we detected ∼5% of dPGS-AuNPs in the cytoplasm, a level undetectable by confocal light microscopy. We also observed ∼5% of dPGS-AuNPs in a rarely identified subcellular site, namely, lipid droplets, which are important for cellular energy metabolism. Finally, we also found substantial changes in the quantity of cytoplasmic organelles upon dPGS-AuNP uptake over the 1–6 h incubation period; the number of small vesicles and mitochondria significantly increased, and the number of multivesicular bodies and the number and volume of lipid droplets significantly decreased. Although nearly all organelle numbers at 6 h were still significantly different from controls, most appeared to be returning to normal levels. To test for generality, we also examined cells after uptake of gold nanoparticles coated with a different agent, polyethylenimine (PEI), used for nucleic acid delivery. PEI nanoparticles did not enter lipid droplets, but they induced similar, albeit less pronounced, changes in the quantity of cytoplasmic organelles. We confirmed these changes in organelle quantities for both nanoparticle coatings by confocal fluorescence microscopy. We suggest this cytoplasmic remodeling could reflect a more common cellular response to coated gold nanoparticle uptake.
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 LCMS/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.
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.
In Deutschland fallen mittelfristig erhebliche Mengen von Mauerwerkbruch an. Aus dem jährlichen Bauschuttanfall und den Produktionszahlen von Wandbausteinen lassen sich die Mengen an Mauerwerksrestmassen ableiten, die als Rückbaumaterial bei Abriss-, Umbau und Sanierungsarbeiten in Deutschland anfallen werden. Hiernach ist pro Jahr mit bis zu 10 Millionen Tonnen heute vornehmlich heterogen anfallendem Mauerwerkbruch zu rechnen [1a]. Im Sinne einer nachhaltigen Kreislaufwirtschaft sind diese Restmassen zukünftig einem möglichst hochwertigen Recycling zuzuführen. In diesem Spannungsfeld führt die deutsche Mauersteinindustrie das breit aufgestellte gemeinschaftliche Forschungsprojekt "SIM Stoffkreislauf im Mauerwerksbau" durch. In insgesamt acht mauersteinübergreifenden IGFForschungsvorhaben wurden und werden technisch orientierte Lösungsmöglichkeiten für das Recycling von Mauerwerk erarbeitet und Anwendungsbereiche für Mauerwerksrestmassen aufgezeigt. Eine grundsätzliche Entscheidung über die Eignung von Mauerwerksrestmassen für bestimmte Recyclingwege kann es letztlich nicht geben. Es wird immer eine Einzelfallentscheidung bleiben, die wesentlich durch komplexe technische sowie ökologische und ökonomische Randbedingungen geprägt wird.
Vor diesem Hintergrund wurde im Rahmen des IGF-Forschungsvorhabens 17022 N der Forschungsvereinigung Porenbetonindustrie e.V., Berlin die Nachhaltigkeit verschiedener Recyclingoptionen für Mauerwerkbruch ganzheitlich untersucht und bewertet. Unter Einbeziehung der Hauptwandbausteinarten Ziegel, Kalksandstein, Porenbeton und Leichtbeton wurden dabei material- und verfahrenstechnische sowie ökologische, ökonomische und soziale Aspekte berücksichtigt. Auf Grundlage der gewonnenen Erkenntnisse wurde ein EDV-gestütztes Analysetool (Web-Tool) zur Nachhaltigkeitsbewertung für das Mauerwerksrecycling entwickelt. Mit dem Web-Tool sollen Anwender (Planer, Architekten, Recycler) die Möglichkeit erhalten, für verschiedene Untersuchungsfälle unter Einbeziehung regional und sachlich bedingter Parameter, wie Materialqualität, Stoffmengen, Logistik, Aufbereitungstechnik, Anwendungsbereiche, Kosten und Erlöse, objektspezifische Aussagen zur Nachhaltigkeit treffen zu können. Auf diesem Wege können im Sinne der aktuellen gesetzlichen Vorgaben nachhaltige Stoffkreisläufe im Mauerwerksbau geschlossen und somit letztlich die Deponierung von Mauerwerksrestmassen vermieden werden.
Alle vier Wandbaustoffarten haben sich im Rahmen des Forschungsvorhabens als recyclingfähig und hinsichtlich der Nachhaltigkeit als gleichwertig erwiesen. Das Ziel des Vorhabens wurde damit erreicht.