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Eingeladener Vortrag
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Carbamazepine (CBZ), an antiepileptic drug, is one of the most commonly detected pharmaceutical drugs in aquatic ecosystems, and is used as a marker of urban pollution. Since CBZ is designed to exert a biological effect, when it reaches aquatic environment high probability exist for toxic effects on non-target organisms. The present study evaluated the acute toxicity of environmentally relevant concentrations of CBZ (0.00, 0.03, 0.30, 3.00, 9.00 µg/L) in the edible clams Venerupis decussata (a native species) and Venerupis philippinarum (an invasive species) collected from the Ria de Aveiro. The effects on both species were assessed through the use of a battery of biomarkers mainly related with health status and oxidative stress. Furthermore, in this work an alternative and promising tool, the direct competitive immunoassay ELISA, for the direct CBZ quantification in clam's tissues, was applied.
The results of the present work showed that CBZ in clam's tissues increased with the exposure concentration and V. decussata gave slightly higher values than V. philippinarum. Although the clams accumulated lower levels of CBZ than the concentration of exposure, these concentrations were enough to impair the health status and induce oxidative stress. However, a different response to CBZ was observed in the two species. While in V. philippinarum the lipid peroxidation levels increased at the highest CBZ concentration (9.00 µg/L), in V. decussata a significant decrease was seen. Moreover, glutathionse S-transferase activity was stimulated in V. decussata and decreased in V. philippinarum. Nevertheless, an induction of glutathione reductase, superoxide dismutase and cytochrome P450 3A4 activities was found in both species as a result of the exposure. The results indicate that, probably, V. philippinarum have a less efficient antioxidant system than V. decussata, and are therefore less capable to neutralize oxidative stress and consequently more sensitive to CBZ. The risk quotient determined for the Ria de Aveiro was higher than 1 indicating that a ecotoxicological risk is suspected. Furthermore, bioaccumulation of CBZ in clams should be taken into consideration since this chemical might be transferred along the food chain and affect non-target organisms.
A novel method that optimizes the screening for antibody-secreting hapten-specific hybridoma cells by using flow cytometry is described. Cell clones specific for five different haptens were analyzed. We selectively double stained and analyzed fixed hybridoma cells with fluorophore-labeled haptens to demonstrate the target-selectivity, and with a fluorophore-labeled anti-mouse IgG antibody to characterize the level of surface expression of membrane-bound IgGs. ELISA measurements with the supernatants of the individual hybridoma clones revealed that antibodies from those cells, which showed the highest fluorescence intensities in the flow cytometric analysis, also displayed the highest affinities for the target antigens. The fluorescence intensity of antibody-producing cells corresponded well with the produced antibodies' affinities toward their respective antigens. Immunohistochemical staining verified the successful double labeling of the cells. Our method makes it possible to perform a high-throughput screening for hybridoma cells, which have both an adequate IgG production rate and a high target affinity.
Homogeneous fluorescence polarization immunoassays (FPIAs) were developed and compared for the determination of caffeine in beverages and cosmetics. FPIAs were performed in cuvettes in a spectrometer for kinetic FP measurements as well as in microtiter plates (MTPs) on a multimode reader. Both FPIAs showed measurement ranges in the µg/L range and were performed within 2 and 20 min, respectively. For the application on real samples, high coefficients of variations (CVs) were observed for the performance in MTPs; the CVs for FPIAs in cuvettes were below 4%. The correlations between this method and reference methods were satisfying. The sensitivity was sufficient for all tested samples including decaffeinated coffee without preconcentration steps. The FPIA in cuvettes allows a fast, precise, and automated quantitative analysis of caffeine in consumer products, whereas FPIAs in MTPs are suitable for semiquantitative high-throughput screenings. Moreover, specific quality criteria for heterogeneous assays were applied to homogeneous immunoassays.
Estrogens, such as 17β-estradiol (E2) and 17α-ethinylestradiol (EE2), are the major responsible for endocrine-disrupting effects observed in aquatic environments due to their high estrogenic potency, even at concentrations ranging from pg L-1 to ng L-1. Thus, it is essential to develop analytical methodologies suitable for monitoring their presence in water samples. Dispersive liquidliquid microextraction (DLLME) was used as a pre-concentration step prior to the quantification of E2 and EE2 by enzyme-linked immunosorbent assay (ELISA). First, an evaluation of the effect of DDLME on the E2 and EE2 ELISA calibration curves was performed. Since the extraction procedure itself had an influence on the ELISA optical density (OD), it became necessary to subject, not only the samples, but also all the standards to the DLLME process. Working ranges were determined, being between 1.2 and 8000 ng L-1, for E2, and between 0.22 and 1500 ng L-1, for EE2. The influence of organic matter, both in the extraction and quantification, was evaluated and it was observed that its presence in the solution did not affect considerably the calibration curve. Recovery rates were also determined, ranging from 77% to 106% for ultrapure water and from 104% to 115% for waste water samples, the most complex ones in what concerns matrix effects. Results obtained when applying the proposed method to real water samples can be considered quite satisfying. Moreover, the obtained working ranges encompass values generally reported in literature, confirming the practical use of the method for environmental samples.
The occurrence of carbamazepine (CBZ) and its metabolites in German and Portuguese wastewater was investigated. A total of 46 samples from influent and effluent wastewater were analyzed by liquid-chromatography (LC) tandem mass spectrometry. The five metabolites 10,11-dihydro-10,11-dihydroxy-CBZ (DiOH-CBZ), 10,11-dihydro-10-hydroxy-CBZ (10-OH-CBZ), 10,11-epoxy-10,11-dihydro-CBZ, 2-hydroxy-CBZ and 3-hydroxy-CBZ were very persistent with little to no removal during wastewater treatment. The highest concentrations were found for CBZ, DiOH-CBZ, and 10-OH-CBZ, with up to 5.0, 4.8 and 1.1 µg/L, respectively. Furthermore, the related pharmaceutical oxcarbazepine and the metabolites 9-hydroxymethyl-10-carbamoylacridan, 1-hydroxy-CBZ (1-OH-CBZ) and 4-hydroxy-CBZ (4-OH-CBZ) were detected. Explicit care was taken to achieve a good chromatographic separation of the numerous isomers that were difficult to distinguish by mass spectrometry alone. A phenylether stationary phase provided the best separation. In combination with high resolution mass spectrometry and hydrogen-deuterium exchange, this LC column enabled us to identify 1-OH-CBZ and 4-OH-CBZ in wastewater for the first time.
The synthesis of a novel PS conjugated with bovine and human serum albumin (BSA and HSA) and a monoclonal antibody anti-CD104 is reported, as well as their biological potential against the human bladder cancer cell line UM-UC-3. No photodynamic effect was detected when the non-conjugated porphyrin was used. Yet, when it was coupled covalently with the mAb anti-CD104, BSA and HSA, the resulting photosensitizer conjugates demonstrated high efficacy in destroying the cancer cells, the mAb anti-CD104 efficacy overruling the albumins.
This study comprises the development of an enzyme-linked immunosorbent assay (ELISA) for the quantification of caffeine in complex aqueous matrices without any sample clean-up procedure. Salinity and dissolved organic matter were selected as potential interfering agents. The addition of a sample buffer containing bovine serum albumin (BSA) prior to the sample was found to decrease the influence of those interfering agents. The working range of the developed method was 0.1–100 µg L- 1. Quantification of caffeine was possible in 43 out of 51 real aqueous samples, at values between < LOD and 15 µg L- 1. Results correlate well with those obtained by LCMS/MS. To the best of our knowledge this is the first study dealing with the quantification of caffeine in Portugal's surface waters.