TY - THES A1 - Oberleitner, Lidia T1 - Immunochemical determination of caffeine and carbamazepine in complex matrices using fluorescence polarization N2 - Pharmacologically active compounds are omnipresent in contemporary daily life, in our food and in our environment. The fast and easy quantification of those substances is becoming a subject of global importance. The fluorescence polarization immunoassay (FPIA) is a homogeneous mix-and-read format and a suitable tool for this purpose that offers a high sample throughput. Yet, the applicability to complex matrices can be limited by possible interaction of matrix compounds with antibodies or tracer. Caffeine is one of the most frequently consumed pharmacologically active compounds and is present in a large variety of consumer products, including beverages and cosmetics. Adverse health effects of high caffeine concentrations especially for pregnant women are under discussion. Therefore, and due to legal regulations, caffeine should be monitored. Automated FPIA measurements enabled the precise and accurate quantification of caffeine in beverages and cosmetics within 2 min. Samples could be highly diluted before analysis due to high assay sensitivity in the low μg/L range. Therefore, no matrix effects were observed. The antiepileptic drug carbamazepine (CBZ) is discussed as a marker for the elimination efficiency of wastewater treatment plants and the dispersion of their respective effluents in surface water. The development of a FPIA for CBZ included the synthesis and evaluation of different tracers. Using the optimum tracer CBZ-triglycine-5-(aminoacetamido) fluorescein, CBZ concentrations in surface waters could be measured on different platforms: one sample within 4 min in tubes or 24 samples within 20 min on microtiter plates (MTPs). For this study, a commercially available antibody was used, which led to overestimations with recovery rates up to 140% due to high cross-reactivities towards CBZ metabolites and other pharmaceuticals. For more accurate CBZ determination, a new monoclonal antibody was produced. In this attempt, methods for improving the monitoring during the production process were successfully applied, including feces screening and cell culture supernatant screening with FPIA. The new monoclonal antibody is highly specific for CBZ and showed mostly negligible cross-reactivities towards environmentally relevant compounds. Measurements at non-equilibrium state improved the sensitivity and selectivity of the developed FPIA due to slow binding kinetics of the new antibody. Additionally, this measure enables for CBZ determination over a measurement range of almost three orders of magnitude. The comprehensively characterized antibody was successfully applied for the development of sensitive homogeneous and heterogeneous immunoassays. The new antibody made the development of an on-site measurement system for the determination of CBZ in wastewater possible. After comprehensive optimization, this automated FPIA platform allows the precise quantification of CBZ in wastewater samples only pre-treated by filtration within 16 min. Recovery rates of 61 to 104% were observed. Measurements in the low μg/L range are possible without the application of tedious sample preparation techniques. Different FPIA platforms including MTPs, cuvettes and tubes were successfully applied. For the choice of the right format, the application field should be considered, e.g. desired sample throughput, usage for optimization or characterization of antibodies or if a set-up for routine measurements is sought for. For high sample throughput and optimization, FPIA performance on MTPs is advantageous. The best results for the application to real samples were obtained using kinetic FP measurements in cuvettes. T3 - BAM Dissertationsreihe - 154 KW - Antibody KW - Coffee KW - ELISA KW - Fluorophore tracer KW - Wastewater PY - 2016 UR - https://nbn-resolving.org/urn:nbn:de:kobv:b43-392506 SN - 978-3-9818270-2-6 SN - 1613-4249 VL - 154 SP - 1 EP - 124 PB - Bundesanstalt für Materialforschung und -prüfung (BAM) CY - Berlin AN - OPUS4-39250 LA - eng AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER - TY - JOUR A1 - Oberleitner, Lidia A1 - Garbe, L.-A. A1 - Dahmen-Levison, Ursula A1 - Schneider, Rudolf T1 - Improved strategies for selection and characterization of new monoclonal anticarbamazepine antibodies during the screening process using feces and fluorescence polarization immunoassay JF - Analytical Methods N2 - Immunoassays are suitable tools for high-throughput screenings. The prerequisite for accurate determinations by these methods is the selection of an excellent antibody. The production and selection of monoclonal antibodies is usually a tedious process. In this study, new strategies for improving antibody production and characterization were applied. This includes the monitoring of the immunization progress in mice through antibodies extracted from feces, which allows a time-resolved and animal-friendly monitoring of the immune response. Additionally, fluorescence polarization immunoassay (FPIA) could be successfully applied for fast and easy examination of cell culture supernatants and the investigation of antibody/antigen interactions including kinetics and fluorescence properties. These methods simplify the selection of the optimal antibody. As a target analyte, carbamazepine was chosen. This is a widely used antiepileptic drug which also frequently occurs in the environment. The new antibody enables CBZ determination in the concentration range 0.66–110 µg L-1 within 10 min using a high-throughput microtiter plate-based FPIA, and between 1.4 and 79 µg L-1 within 5 min applying an automated cuvette-based FPIA instrument, and from 0.05–36 µg L-1 using ELISA. The measurements were performed at a non-equilibrium state which improved the sensitivity and selectivity of the assays. Due to low cross-reactivity especially towards the main CBZ metabolite and other pharmaceuticals (<1%), this antibody gives the opportunity for application in medical and environmental analyses. KW - Antikörper KW - Emerging Pollutants KW - Schadstoffe KW - Monoklonal KW - Pharmazeutika KW - Arzneistoffe KW - Assay KW - Pesticide parathion-methyl KW - Waste-water KW - Samples KW - Pharmaceuticals KW - Quantification PY - 2016 UR - https://nbn-resolving.org/urn:nbn:de:kobv:b43-392473 DO - https://doi.org/10.1039/c6ay01968d SN - 1759-9660 VL - 8 SP - 6883 EP - 6894 PB - Royal Society of Chemistry CY - London AN - OPUS4-39247 LA - eng AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER -