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Partikelbasierte Multianalyt- Fluoreszenzimmunoassays für die Unweltanalytik und Biotechnologie
(2021)
Zur Beurteilung der Wasserqualität können Markersubstanzen, wie Carbamazepin, Diclofenac, Koffein und Gallensäuren, verwendet werden. Erhöhte Konzentrationen dieser anthropogenen Marker weisen auf Einleitstellen von Abwasser hin und zeigen somit an, wo Gewässer verschmutzt werden. Diese Arbeit beschreibt deshalb die Etablierung, Optimierung und Validierung eines partikelbasierten Suspensionsarray Fluoreszenzimmunoassays (SAFIA), mit dem diese Substanzen gleichzeitig und parallel in vielen Proben quantitativ bestimmt werden können. Für SAFIA wurde das Format des kompetitiven indirekten Immunoassays gewählt. Als Plattform wurden fluoreszenzintensitätscodierte Polystyrol-Kern/Siliziumdioxid-Schale-Partikel, auf deren Oberfläche Haptene als kompetitive Bindungsstellen für Antikörper immobilisiert wurden, verwendet. Hier zeigte sich, dass eine selektive Erkennung der Haptenstruktur auf der Oberfläche der Partikel durch die Antikörper nur gegeben war, wenn die Partikel zusätzlich mit Polyethylenglykolgruppen funktionalisiert wurden. Zum Auslesen des Fluoreszenzsignals wurde ein Durchflusszytometer verwendet. Kompatibilität mit der Durchführung in Mikrotiterplatten wurde erreicht, indem eine Stopp-lösung für den SAFIA, basierend auf Formaldehyd, entwickelt wurde. Nach der Optimierung der Assayparameter können die oben genannten vier Analyten gleichzeitig in einer Probe mindestens bis zu einer Konzentration von 0.3 μg L-1 nachgewiesen werden. Der Assay kann ohne Wasch-schritte ausgeführt werden und ist somit einfacher als konventionelle Immunoassays, wie z. B. ELISA. Der SAFIA wurde hinsichtlich Interferenzen und Selektivität untersucht. Dabei zeigte sich, dass Matrixbestandteile, die in wässrigen Umweltproben vorkommen, keinen bzw. nur einen ge-ringen Einfluss auf den SAFIA hatten. Im simulierten Umweltscreening eines Flusses und mithilfe der Analyse von Abwasserproben wurde SAFIA validiert; hier zeigte SAFIA eine mit ELISA vergleich-bare Genauigkeit, bei gleichzeitiger Senkung der Analysenzeit und -kosten. Die gleichzeitige Detektion mehrerer anthropogener Marker erlaubte zudem Rückschlüsse auf die Art von Verschmutzungsquellen, im Gegensatz zu ELISA, mit dem nur ein Analyt bestimmt werden kann. Da hochaffine Antikörper für Immunoassays essenziell sind, wurde ein SAFIA zum Screening antikörperproduzierenden Zellen (Hybridomzellen) entwickelt und eingesetzt. Durch die Implementierung eines Sandwichimmunoassays zur IgG Bestimmung und durch Verwendung von homo- und heterologen Haptenstrukturen konnten aus einem Pool von Hybridomzellen diejenigen ausgewählt werden, deren sekretierter Antikörper die geringsten Nachweisgrenzen im Immunoassay erlauben. Damit konnte das Anwendungsspektrum des SAFIA maßgeblich vergrößert werden. Er vereinfacht als biotechnologisches Werkzeug stark sowohl die Produktion von monoklonalen Antikörpern als auch die Analyse von Umweltproben.
A bead suspension array for measuring emerging pollutants and anthropogenic markers in wastewater.
(2016)
The assessment of water quality demands for high-throughput and multi-target compatible analytical methods. On the one hand, the influence of sewage on natural surface waters must be routinely monitored - according to the European Water Framework Directive. On the other hand, estimation of removal efficiencies of pollutants, such as drug residues, is in the focus of industrial and public wastewater treatment. Immunoassays, such as ELISA, are often applied in this area owing to their cost-efficiency and high-throughput capability. However, it is only possible to measure a single analyte in one measurement.
In contrast, array technology is capable for measuring multiple substances in parallel. Here we present a four-plex bead-based flow cytometric assay for measuring three drugs (carbamazepine, an anti-epileptic drug, diclofenac, an antiphlogistic and caffeine, a psychoactive substance) and the bile acid isolithocholic acid, which is proposed to be used as a fecal marker.
In this suspension array core-shell particles, consisting of a polystyrene core and a silica shell are used. They can be easily encoded with a fluorophore which is introduced to the core and functionalized with amino groups on the surface for creating binding sites for antibodies: Therefore, the marker substances are bound to the amino groups via NHS chemistry.
For the determination of the pollutants, the beads are incubated with a mixture of the analyte-specific antibodies. The antibodies undergo a competitive reaction to bind the immobilised molecules on the surface of the beads or the free analyte in the solution, respectively. Bound antibodies can then be visualized via fluorophore-labelled secondary antibodies. For read-out a flow cytometer is used to virtually separate the different encoded beads and to measure the resulting signal intensity of the immunoassay. In order to obtain highly selective binding of the antibodies, we investigated different types of surface modifications to overcome unspecific binding, finding that a PEG-based surface is suitable to support our immunoassay format. The resulting multiplexing assay is appropriate to detect the marker substances in the low µg/L range.
Supernatants from a fermentation process of Pichia pastoris were investigated by Raman spectroscopy. Using partial least squares regression, the principal substrates glycerol and methanol could be predicted, however not the expressed protein. To gain further insight, a priori prepared calibration samples were studied by vibrational-, UV/Vis-, and fluorescence spectroscopy. For the quantification of glycerol and methanol, Raman spectroscopy was identified as the most sensitive technique, and superior to near-infrared spectroscopy, but not for protein contents below 1 g L–1. Both UV/Vis absorption and fluorescence spectroscopy are well suited for the quantification of protein, however, best results were obtained with UV/Vis absorption.
The calibration of fluorescence signals in assays as well as in biological systems is a key requirement. Especially for quantitative studies of living cells, e.g. expression of biomolecules the generation of concentration-proportional analytical statements obtained by the fluorescence intensity plays a significant role. This also applies for cell assays like immunofluorescence assays. Also, the determination of local concentrations of target proteins within cells or within their environment is a major challenge in modern biology.
Proper calibration of the fluorescence imaging systems is the prerequisite to ensure that test results from different instruments and different test environments are comparable. The existing calibration solutions for fluorescence imaging systems, however, mostly depend on simple instrument calibration without reference to absolute physical standards or with insufficient stability for multiple measurements. In order to obtain the necessary quantitative information, it is required to calibrate both instruments and the corresponding assays, ideally with one calibrating system.
We present several new calibration tools for the most commonly used platforms for cytometry, i.e. fluorescence microscope based systems and flow cytometry based systems. They are comprised of ultra-stable fluorophores that are encapsulated in bio- and cell-compatible polymer matrices. Each tool is tailored to the specific requirements of the different read-out platforms. Both re-usable single-color calibration slides for fluorescence microscopes as well as new multi-color calibration beads are presented.
The traceability of the calibration tools to international standards and the wide calibration range of the fluorescence intensity are illustrated. They are thus suitable for calibration of the signal over concentration and quantum yield to that of the targets.
Zur Onlineüberwachung von Fermentationsprozessen werden zunehmend schwingungsspektroskopische (Raman-, Mittel- und Nahinfrarotspektroskopie (NIRS) und UV-/VIS- Methoden(Absorptions- und Fluoreszenzspektroskopie) in Kombination mit multivariater Auswertung eingesetzt. Anliegen dieser Arbeit war es, zu testen, welche Verfahren für die spektroskopische On- und Offlineüberwachung des Fermentationsprozesses zur biotechnologischen Herstellung einer Malariavaccine grundlegend geeignet sind und welche Messbereiche und Genauigkeiten der Vorhersage im Idealfall bei der Abwesenheit von Hefezellen erzielt werden können.
This short course offers an introduction in immunomicroarrays and its application in environmental, food and clinical analysis. Basic principles of microarray fabrication, including spotting techniques and immobilization chemistry are presented, as well as different immunoassay formats and data treatment strategies. Furthermore, principles of bead-based suspension arrays are introduced, including principles of ligand and receptor immobilization, encoding and read-out strategies.
The lab-on-valve (LOV) is a mesofluidic platform that has been recently exploited for
the automation and miniaturization of bioanalytical assays, resorting namely to
molecular recognition schemes based on immunosensing. Due to its high versatility
for reagent accommodation, it is possible to establish immunoassays under several
formats (eg. direct competitive ELISA, sandwich ELISA or even label-free immunoaffinity
chromatography). For instance, the LOV has been used as a manifold for
UV-vis micro-Bead Injection Spectroscopy (μ-BIS), a technique that involves the
quantification of the target analyte by direct measurement on the surface of a solid
phase capable of retaining the target analyte by molecular recognition.
The μ-BIS-LOV strategy affords several analytical advantages, namely short time-toresult
intervals (3 to 15 min), low sample volume (1-20 μL), automated solution handling
and washing steps, downscaling of reagents’ consumption, low-cost analysis
and little generation of waste. Additionally, the solid support is renewed before each
determination, minimizing surface fouling, cross-contamination issues and functional
group deactivation. No sample clean-up steps are required because interferences
are separated from the target analyte upon quantification mediated by a molecular
recognition element attached to the micro-bead column. The portability of the LOV
device makes it compatible with point-of-care testing.
To our knowledge, this technique has been mainly employed for the evaluation and
optimization of bioaffinity processes, but its potential for clinical and environmental
analysis remains underexploited. Hence, in this communication, different immunosensing
strategies using the LOV platform will be addressed, namely the determination
of autoimmune IgG in human serum, and the assessment of drug (carbamazepine)
levels in wastewater samples.
The conventional hybridoma screening and subcloning process is generally considered to be one of the most critical steps in hapten-specific antibody production. It is time-consuming, monoclonality is not guaranteed, and the number of clones that can be screened is limited. Our approach employs a novel hapten-specific labeling technique of hybridoma cells. This allows for fluorescence-activated cell sorting (FACS) and single-cell deposition and thereby eliminates the above-mentioned problems. A two-step staining approach is used to detect antigen specificity and antibody expression: in order to detect antigen specificity, hybridoma cells are incubated with a hapten−horseradish peroxidase conjugate (hapten−HRP), which is subsequently incubated with a fluorophore-labeled polyclonal anti-peroxidase antibody (anti-HRP−Alexa Fluor 488). To characterize the expression of membrane-bound immunoglobulin G (IgG), a fluorophore-labeled anti-mouse IgG antibody (anti-IgG−Alexa Fluor 647) is used. Hundreds of labeled hybridoma cells producing monoclonal antibodies (mAbs) specific for a hapten were rapidly isolated and deposited from a fusion mixture as single-cell clones via FACS. Enzyme-linked immunosorbent assay (ELISA) measurements of the supernatants of the sorted hybridoma clones revealed that all hapten-specific hybridoma clones secrete antibodies against the target. There are significant improvements using this high-throughput technique for the generation of mAbs including increased yield of antibody-producing hybridoma clones, ensured monoclonality of sorted cells, and reduced development times.
