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Eingeladener Vortrag
- nein (46)
Diclofenac (DCF) is a widely used drug against fever, inflammation, pain, and rheumatic diseases. An average of 70 % of the ingested diclofenac is excreted in the urine. Thus, 63 tons per year are introduced into the water cycle in Germany. Due to insufficient removal of diclofenac in wastewater treatment plants, residues of diclofenac can be found in surface water and sometimes in drinking water. In order to establish an immunoassay, an anti-DCF antibody was required. Some antibody developments have been reported, based on an immunogen resulting from direct coupling of diclofenac to proteins via the carboxylic function. Finally, we used a monoclonal antibody, clone F01G21.
The talk introduces the technique of LC-ELISA: fractionating pre-concentrated water samples, analyzing them for containing compounds that bind to hapten-selective antibodies followed by high-Resolution mass spectrometry non-target analysis of "positive" fractions for Transformation products of emerging organic contaminants.
Im Beitrag wurden Ergebnisse von Elutionsuntersuchungen mit PFC kontaminierten Bodenmaterialien vorgestellt. Zum einen waren das Untersuchungen zur Eignung von Säulenversuchen für diese Schadstoffgruppe und zum anderen vergleichende Schüttel- und Säulenversuche für zwei verschiedene Böden mit unterschiedlichen PFC-Kontaminationsquellen.
A vast number of emerging pollutants has been detected in the environment over the last decades. Analytical methods suitable for trace analysis are needed that are desirably also fast, inexpensive and, if possible, robust and portable. Immunoanalytical methods which are available in a broad range of formats, can be profitably used here to analyze for the distribution and the trends of concentration levels of contaminants in the environment. Some of these formats are single-analyte but high-throughput methods. In order to use them wisely, indicator substances, sometimes called anthropogenic markers, should be selected and used in screening approaches. Other methods are suitable to be performed on portable instrumentation in the field (on-site) or in facilities such as wastewater treatment plants for on-line monitoring. Furthermore, there are the socalled array technologies that allow for parallel analysis of several analytes of interest (multiplexing). The microtiter-plate based ELISA (Enzyme-linked Immunosorbent Assay) is the method of choice for the analysis of a large number of samples [1]. ELISA screening data for anthropogenic markers such as the antiepileptic carbamazepine, the analgesic diclofenac, the anti-histaminic cetirizine, the steroid hormone estrone, the antimicrobial sulfamethoxazole, the stimulants caffeine and cocaine, the priority pollutant bisphenol A, and the bile acid isolithocholic acid [2] are presented. For on-site screening and monitoring, simpler formats, like mix-and-read assays, e.g. the Fluorescence Polarization Immunoassay (FPIA) or Lateral-flow Immunoassays (LFIA) are more suitable tools. Electrochemical formats run on portable devices provide additional advantages as no light source is required. Some examples are presented and discussed. The suitability of multi-analyte formats such as immunomicroarrays depends on the choice of a signal-producing system that provides small uncertainties and good reproducibility of the measurements. Biochip (“flat”) arrays read out on slide scanners and bead-based (“suspension”) arrays read out in flow cytometers are two options and show their distinct pros and cons. Altogether these approaches show the great potential immunoanalytical methods provide for the screening for environmental contaminants in the aquatic environment.
Monitoring water quality regarding emerging pollutants, such as drug residues, demands for selective, high-throughput and multi-target analytical methods. On the one hand, the influence of sewage on natural surface waters must be routinely monitored. 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, offer the possibility to be highly sensitive and selective due to the high target affinity and specificity of target recognition by antibodies. Batch-wise processing in microtiter plates allows for the necessary high-throughput, but only a single analyte can be determined within one measurement.
To overcome these disadvantages, we developed a four-plex Suspension Array Fluorescence Immunoassay (SAFIA), which is adaptable for the microtiter plate format. The modular and self-prepared bead support consists of polystyrene-core silica-shell microparticles. While the polystyrene core is used for encoding, by introducing different amounts of fluorescent dyes, the silica shell creates a solid-support for the immunoassay: the target analytes, three drugs, carbamazepine, diclofenac and caffeine, and the fecal marker isolithocholic acid are covalently coupled to amino groups on the surface via NHS chemistry. Specific recognition of these haptens is accomplished by further introduction of PEG moieties, suppressing non-specific binding. A competitive immunoassay is subsequently conducted in a simple mix-and-read procedure, eliminating the demand for laborious washing steps and decreasing time-to-result. An automated flow cytometer is used for simultaneous decoding and quantification.
After optimization, the SAFIA showed limits of detection for all analytes in the low µg L 1 range, meeting the sensitivity criteria for wastewater analysis. Then, applicability of the SAFIA was studied on real wastewater samples from three different wastewater treatment plants in Berlin. The results were in good comparability to LC-MS/MS indicating high matrix stability. Moreover, the accuracy of the assay exceeded that of the respective ELISA. Finally, we used SAFIA to assess the influent of treated and untreated wastewater on the Douro river estuary in Portugal. The obtained results of the analysis were comparable to ELISA. However, the measurements by SAFIA could be carried out in one quarter the time of analysis required for ELISA.
Acknowledgements: The authors acknowledge financial support from Deutscher Akademischer Austauschdienst and from Fundação das Universidades Portuguesas under the protocol CRUP-DAAD (Ações Integradas Luso-Alemãs nºE-20/16, DAAD Zuwendungsvertrag 57212899).