Biomedical subjects

Reinhard Niessner

Publications and source records attributed to Reinhard Niessner.

At least 19 recordsLinked to original sources

PubMed2005

Sandwich immunoassays for the determination of peanut and hazelnut traces in foods.

People suffering from food allergies are dependent on accurate food labeling, as an avoidance diet is the only effective countermeasure. Even a small amount of allergenic protein can trigger severe reactions in highly sensitized patients. Therefore, sensitive and reliable tests are needed to detect potential cross-contamination. In this paper two fast sandwich immunoassays are described for the determination of peanut (Arachis hypogaea) and hazelnut (Corylus avellana) traces in complex food matrices. Mouse monoclonal antibodies were used as capture antibodies, and labeled rabbit polyclonal antibodies were used as detection antibodies in both assays. The assay time was 30 min in total, and cross-reactivities against a variety of fruits and seeds were found to be in the low 10(-4)% (ppm) level or in some cases not detectable. The recoveries in all tested food matrices ranged from 86 to 127%, and the limits of detection were in the range of 0.2-1.2 mg/kg (ppm) in food for both peanut and hazelnut, respectively.

Allergens↗

PubMed2005

Protein nitration by polluted air.

The effects of air pollution on allergic diseases are not yetwell-understood. Here, we show that proteins, in particular birch pollen proteins including the allergen Bet v 1, are efficiently nitrated by polluted air. This posttranslational modification of proteins is likely to trigger immune reactions and provides a molecular rationale for the promotion of allergies bytraffic-related air pollution. Enzyme immunoassays have been used to determine equivalent degrees of nitration (EDN) for protein samples exposed to urban outdoor air and synthetic gas mixtures. The observed rates of nitration were governed by the abundance of nitrogen oxides and ozone, and concentration levels typical for summer smog conditions led to substantial nitration within a few hours to days (EDN up to 20%). Moreover, nitrated proteins were detected in urban road dust, window dust, and fine air particulate matter (EDN up to 0.1%).

Air Pollutants↗

PubMed2004

Development of a class-specific ELISA for sulfonylurea herbicides (sulfuron screen).

The development of a direct competitive ELISA for the detection of a broad range of sulfonylurea herbicides (SUs) is described. Polyclonal antibodies were generated in rabbits using three different immunizing haptens. Antiserum with the broadest specificity was obtained with a mesosulfuronbenzylamine derivative which was coupled via a succinic acid spacer to keyhole limpet hemocyanine. A heterologous enzyme tracer which did not contain the succinic acid bridge was prepared using activated horseradish peroxidase. The direct competitive ELISA was optimized and applied for spiked tap and surface water samples. From 30 SUs, 8 compounds showed a molar cross-reactivity (CR) higher than 100% (this value was set for the hapten) and 11 compounds CRs between 10% and 100%. The ELISA can detect 16 SUs at a concentration of 0.1 microg/L or lower. Different surface and tap water samples were spiked with chlorimuron ethyl, metsulfuron methyl, or primisulfuron methyl at concentrations of 100, 200, or 500 ng/L and subsequently analyzed by both ELISA and HPLC-UV. Correlation analysis revealed good agreement between both methods (r2 = 0.983/0.948/0.982; n = 21 for each analyte). Using ELISA, no sample pretreatment other than filtration was necessary.

Antibodies↗

PubMed2004

Comparison of nitrotyrosine antibodies and development of immunoassays for the detection of nitrated proteins.

Three monoclonal antibodies (mAb) and three polyclonal antibodies (pAb) have been characterized and compared with respect to their cross-reactivities and affinities for 3-nitrotyrosine, eight aromatic compounds with similar chemical structures, a peptide containing a single nitrotyrosine residue, and fourteen nitrated protein standards (bovine serum albumin, BSA) containing different numbers of nitrotyrosine residues per protein molecule (0.2 to 16.8). In indirect competitive immunoassays, mAb Alexis 39B6 exhibited the highest affinity for free 3-nitrotyrosine (10(6) L mol(-1)), while the pAb Oxis 24312 from sheep exhibited the highest affinities for nitrated proteins (up to 10(8) L mol(-1)). The apparent affinities determined in the indirect competitive assays were inversely correlated with the limits of detection (LOD) determined in one-sided immunoassays. With the sheep pAb minimum LOD on the order of 10 pmol L(-1) were achieved for highly nitrated proteins, corresponding to effective LOD on the order of 100 pmol L(-1) for nitrotyrosine residues. In the one-sided assays, however, the LOD for nitrated proteins increased proportionally with increasing background concentrations of native proteins in the investigated samples. Sandwich immunoassays combining pAb and mAb for selective enrichment and detection of nitrated proteins allowed to eliminate this native protein matrix effect and to achieve LOD on the order of 300 pmol L(-1) for highly nitrated proteins independent of native protein background concentrations.

Air Pollutants↗

PubMed2004

Microanalysis by laser-induced plasma spectroscopy in the vacuum ultraviolet.

A new setup for microanalysis by laser-induced plasma spectroscopy in the VUV range is presented and described in detail. The system features an integrated ablation and detection module with a newly designed VUV echelle system. The echelle permits a full spectral coverage between 150 and 300 nm with a resolving power lambda/Deltalambda between 11,000 and 15,000. At present, the ablation module permits a microanalysis with a crater size of 25 microm and a nominal depth resolution with an ablation rate of 150 nm/pulse. The VUV performance was demonstrated for bulk analysis of steel; detection limits for sulfur, carbon, and phosphorus were in the lower milligram per kilogram range. The VUV scanning and mapping performance for heterogeneous matrixes was illustrated for mineral bottom ash samples from a waste incineration process.

Journal Article↗

PubMed2004

Automated microarray system for the simultaneous detection of antibiotics in milk.

A parallel affinity sensor array (PASA) for the rapid automated analysis of 10 antibiotics in milk is presented, using multianalyte immunoassays with an indirect competitive ELISA format. Microscope glass slides modified with (3-glycidyloxypropyl)trimethoxysilane were used for the preparation of hapten microarrays. Protein conjugates of the haptens were immobilized as spots on disposable chips, which were processed in a flow cell. Monoclonal antibodies against penicillin G, cloxacillin, cephapirin, sulfadiazine, sulfamethazine, streptomycin, gentamicin, neomycin, erythromycin, and tylosin allowed the simultaneous detection of the respective analytes. Antibody binding was detected by a second antibody labeled with horseradish peroxidase generating enhanced chemiluminescence, which was recorded with a sensitive CCD camera. All liquid handling and sample processing was fully automated, and one analysis was carried out in milk within less than 5 min. The detection limits ranged from 0.12 (cephapirin) to 32 microg/L (neomycin). Penicillin G could be detected at the maximum residue limit (MRL); the detection limits for all other analytes were far below the respective MRLs. The PASA system proved to be the first immunochemical biosensor platform having the potential to test for numerous antibiotics in parallel, such being of considerable interest for the control of milk in the dairy industry.

Animals↗

PubMed2003

Analysis of nitrated polycyclic aromatic hydrocarbons by liquid chromatography with fluorescence and mass spectrometry detection: air particulate matter, soot, and reaction product studies.

Polycyclic aromatic hydrocarbons (PAH) and their nitrated derivatives (nitro-PAH) are environmental pollutants which pose a threat to human health even at low concentration levels. In this study, efficient analytical methods for the analysis of nitro-PAH and PAH (extraction, clean-up, chromatographic separation, and spectrometric detection) have been developed, characterized, and applied to aerosol samples. The separation and quantification of 12 nitro-PAH was carried out by reversed-phase high performance liquid chromatography (HPLC), on-line reduction, and fluorescence detection. The detection limits were in the range of 0.03-0.5 microg L(-1) (6-100 pg in the investigated sample aliquots), and the recovery rates from soot samples were 70-90%. Nitro-PAH and PAH concentrations have been determined for different types of soot and for urban, rural, and alpine fine air particulate matter (PM2.5). For the first time, trace amounts of nitro-PAH have been detected in a high-alpine clean air environment. The on-line reduction and fluorescence technique has been complemented by atmospheric pressure chemical ionization time-of-flight mass spectrometry (APCI-TOF-MS). The MS detection allowed the analysis of partially nitrated and oxygenated PAH in laboratory studies of the heterogeneous reaction of PAH on soot and glass fiber substrates with gaseous nitrogen oxides and ozone. It led to the tentative identification of a previously unknown nitrated derivative of the particularly toxic PAH benzo[ a]pyrene (BaP-nitroquinone), and provides the first experimental evidence that PAH-nitroquinones can be formed by reaction of PAH with atmospheric photooxidants.

Journal Article↗

PubMed2003

Development of antibodies for the detection of N-acetyl-glufosinate.

Glufosinate is a widely used herbicide, which is difficult to detect by conventional analytical techniques. For many other herbicides, suitable antibodies have been raised for immunoassay development. Unfortunately, glufosinate is a very small molecule and difficult to immunize with. Thus, a derivatization-assisted immunoassay (DAIA) using the target analyte N-acetyl-glufosinate (NAG) was constructed. The activated hapten was synthesized by a new approach, using a homobifunctional cross-linker suberic acid bis(N-hydroxysuccinimide ester). The preparation of a suitable conjugate, the immunization, and the characterization of polyclonal antibodies are shown. The determination of the conjugation density (hapten density) of the immunogens was performed by four different methods (high-performance liquid chromatography with a refractive index detector, total reflection X-ray fluorescence, inductively coupled plasma mass spectrometry, and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry), which gave similar results. The limit of detection was 17 mug/L NAG in water for the direct competitive enzyme immunoassay. NAG is also a main metabolite of glufosinate in resistant transgenic plants. The antibodies might be useful for the selective detection of NAG in the presence of the parent compound glufosinate (cross-reactivity 0.13%) and other metabolites.

Acetylation↗

PubMed2003

Methods for studying synaptosomal copper release.

Cu is thought to play an important role in the pathogenesis of several neurodegenerative diseases, such as Wilson's, Alzheimer's, and probably in prion protein diseases like Creutzfeld-Jakob's disease. Until now, no method existed to determine the concentration of this cation in vivo. Here, we present two possible approaches combined with a critical comparison of the results. The successful use of fluorescent ligands for the determination of Ca2+-concentrations in recent years encouraged us to seek a fluorophore which specifically reacts to Cu2+ and to characterize it for our purposes. We found that the emission of TSPP (tetrakis-(4-sulfophenyl)porphine) at an emission wavelength of 645 nm is in vitro highly specific to Cu2+ (apparent dissociation constant Kd=0.43 +/- 0.07 microM at pH 7.4). It does not react with the most common divalent cations in the brain, Ca2+ and Mg2+, unlike most of the other dyes examined. In addition, Zn2+ quenches TSPP fluorescence at a different emission wavelength (605 nm) with a Kd of 50 +/- 2.5 microM (pH 7.0). With these findings, we applied the measurement of Cu with TSPP to a biological system, showing for the first time in vivo that there is release of copper by synaptosomes upon depolarisation. Our findings were validated with a completely independent analytical approach based on ICP-MS (inductively-coupled-plasma mass-spectrometry).

Animals↗

PubMed2003

Spatial and temporal observations of adsorption and remobilization of heavy metal ions in a sandy aquifer matrix using Magnetic Resonance Imaging.

The exploration of the transport and matrix interactions of heavy metal ions in the subsurface environment under natural conditions is an important field of research in environmental science and technology. Most commonly, column tests are used for a first assessment of the transport behavior. Classical column tests fall short with regard to the spatial and temporal resolution; however, these detailed data are needed for proper upscaling. Hence, providing spatially and temporally resolved data on the distribution of environmentally relevant concentrations of heavy metal ions in a water-wet aquifer matrix poses a major challenge to analytical chemistry. In this contribution, we present the results of Magnetic Resonance Imaging (MRI) studies in which submilligram quantities of heavy metal ions where either fed conventially through the column or locally injected into saturated sand packings. The subsequent transport and mobilization was monitored at a high spatial and temporal resolution. The results from a local injection show that the test design of column tests has not yet come to an end and that column tests under MRI-control may be used as a model system for, e.g., remediation techniques.

Adsorption↗

PubMed2003

Residue analysis of the pharmaceutical diclofenac in different water types using ELISA and GC-MS.

A highly sensitive and specific indirect competitive enzyme-linked immunosorbent assay (ELISA) for the determination of diclofenac in water samples was developed. With pure water, the limit of detection (LOD, S/N = 3) and IC50 were found to be 6 ng/L and 60 ng/L, respectively. The analytical working range was about 20-400 ng/L. Highest cross-reactivity (CR) of 26 tested pharmaceuticals, metabolites, and pesticides was found for 5-hydroxydiclofenac (100%). Other estimated values were well below 4% and, therefore, are negligible. The assay was applied for the determination of diclofenac in tap and surface water samples as well as wastewater collected at 20 sewage treatment plants (STPs) in Austria and Germany. Humic substances were identified as main interference in surface water. Wastewater samples which were only submitted to filtration and dilution yielded about 25% higher diclofenac concentrations using the ELISA compared to GC-MS. However, the ELISA turned out to be a simple, inexpensive, and accurate method for the determination of diclofenac both in influent and effluent wastewater after rather simple sample preparation, i.e., filtration, acidification, and readjustment to neutral pH-value, and at least 10-fold dilution with pure water.

Anti-Inflammatory Agents, Non-Steroidal↗

PubMed2003

Polycyclic aromatic hydrocarbons in urban air particulate matter: decadal and seasonal trends, chemical degradation, and sampling artifacts.

Aerosol filter samples collected at a major urban traffic junction (LKP) and at a suburban residential location (IWC) in the metropolitan area of Munich (Germany) throughout the years 2001 and 2002 have been analyzed for 12 of the 16 EPA priority polycyclic aromatic hydrocarbon (PAH) pollutants by liquid chromatography with fluorescence detection. The mean mass concentration of the sum of all investigated PAH in the sampled air at LKP (1.9-5.0 ng m(-3)) was roughly two times higher than at IWC (0.8-2.9 ng m(-3)), and at both locations it was about 2-3 times higher in winter (heating season) than in summer and spring or autumn. Comparisons with earlier measurement campaigns indicate a steep decrease of PAH abundance by almost an order of magnitude from 1980 to 1993 and a much slower decrease since then. Distinctly different seasonal trends and short-term fluctuations have been observed for semivolatile 3- and 4-ring PAH and for particle-bound 5- and 6-ring PAH. Based on systematic correlation analyses with a wide range of air quality parameters, most of the differences can be attributed to not only varying emissions but also chemical reactions with atmospheric oxidants which were found to play an important role. The results of denuder experiments prove that substantial degradation of the particularly toxic tracer benzo[a]pyrene and of the other investigated 5- and 6-ring PAH can occur during filter sampling and on airborne particles (formation of oxygenated and nitrated derivatives). Filter reaction artifacts are shown to lead to an underestimation of the actual PAH content of urban air particulate matter by up to 100% of the measurement value or more, with a near-linear dependence on ozone volume mixing ratio. The role and applicability of ozone as a tracer of atmospheric oxidizing capacity for particle-bound PAH is discussed and confirmed by comparison with earlier investigations and by complementary laboratory experiments (reaction kinetics and product studies).

Air Pollutants↗

PubMed2003

Microarrays for the screening of allergen-specific IgE in human serum.

The described in vitro test system for allergy diagnosis is based on microscope glass slides activated with (3-glycidyloxypropyl)trimethoxysilane. Allergen solutions are immobilized as small droplets (approximately 10 nL) on the activated glass slides with a piezoelectric arrayer. In contrast to other tests for specific IgE, such as Pharmacia CAP FEIA, AlaSTAT, or FAST, only a 25-microL serum sample is needed for the screening of allergen-specific IgE against a multitude of allergens and the test can be performed in less than 1 h. Compared with multiallergen dipstick screening tests (e.g., IgEquick, CMG Immunodot) based on multiallergen-coated nitrocellulose strips, the measurement of the microarray-based system can be performed automatically. The chemiluminescence intensities are detected with a sensitive CCD camera. Allergen extracts and recombinant/purified allergens (24 preparations) have been used on the same modified surface for the screening of allergen-specific IgE. With these disposable microarray slides, it is possible to distinguish between patients with and without elevated levels of allergen-specific IgE. Repeated measurements of serum samples demonstrated a sufficient reproducibility. Detection limits (microg/L) of 0.35 (r Betvl), 0.16 (PLA2), and 1.9 (Der p1) were achieved.

Allergens↗

PubMed2003

Oxygen determination in oxygen-supersaturated drinking waters by NMR relaxometry.

In recent years, a rising number of different table waters supersaturated with oxygen have hit the market with claims of both positive health effects and an increase in athletic performance. In addition to research on the correctness of these claims and their possible physiological reasons, the appearance of oxygen supersaturation as a marketing promise also creates a need for appropriate analytical techniques allowing a rapid and reliable determination of oxygen contents in such waters. Here, we present NMR relaxometry as a possible analytical tool for such studies. NMR relaxation in oxygen supersaturated water is not only of interest in the context of monitoring the oxygen content in such drinking water products, but also might offer some interesting possibilities in medical and environmental science MRI applications. As a contact-less measuring method, NMR relaxometry avoids disturbance of the measurement due to outgassing. The method was applied in the concentration range from 10 mg l(-1) to over 100 mg l(-1) dissolved oxygen. In addition to freshly sampled drinking waters, also oxygen losses during storage of the water in open drinking vials was studied.

Beverages↗

PubMed2003

Enzyme immunoassays for the investigation of protein nitration by air pollutants.

Two enzyme immunoassays have been developed, characterised, and applied to investigate protein nitration in birch pollen extract (BPE) and bovine serum albumin (BSA) samples exposed to air pollutants. The monoclonal antibody CAY-189542 against nitrotyrosine (raised against peroxynitrite-treated keyhole limpet hemocyanine) was characterised in an indirect competitive assay (affinity and cross-reactivities) and applied in a new one-sided enzyme immunoassay for nitrated proteins. The one-sided assay was calibrated against a nitrated BSA standard with an average of 14 nitrotyrosine residues per molecule (nitro-(14)-BSA; detection limit 8.3 pmol L(-1)), and the sensitivity of the test was found to be significantly enhanced by a multivalent binding mode of the monoclonal antibody (bonus effect of multivalency). The same antibody and a polyclonal antibody against Bet v 1, the most prominent birch pollen allergen, were used in a new sandwich immunoassay for specific determination of nitrated Bet v 1. This assay was calibrated against a nitrated Bet v 1 standard with an average of 3 nitrotyrosine residues per molecule (nitro-(3)-Bet v 1; detection limit 0.2 nmol L(-1)). Bet v 1 and BSA exposed to polluted urban outdoor air and to synthetic gas mixtures containing NO2 and O3 at atmospherically relevant concentration levels were found to be efficiently nitrated within hours to days. Pronounced correlations of nitro-(14)-BSA equivalent concentrations with exposure time and with nitro-(3)-Bet v 1 equivalent concentrations in nitrated BPE samples were observed. Test experiments indicated that the efficiency of protein nitration was strongly enhanced by reactive species formed upon interaction of NO2 with O3 and H2O (e.g. NO3 and HNO3). Potential implications of protein nitration by air pollutants are outlined and discussed.

Air Pollutants↗

PubMed2002

Selective trace analysis of sulfonylurea herbicides in water and soil samples based on solid-phase extraction using a molecularly imprinted polymer.

A molecularly imprinted polymer (MIP) was synthesized using the herbicide metsulfuron-methyl (MSM) as a template, 2-(trifluoromethyl)acrylic acid as a functional monomer, divinylbenzene as a cross-linker, and dichloromethane as a porogen. This polymer was used as a solid-phase extraction material for the quantitative enrichment of five sulfonylureas (nicosulfuron, thifensulfuron-methyl, metsulfuron-methyl, sulfometuron-methyl, and chlorsulfuron) in natural water and soil samples and off-line coupled to a reversed-phase HPLC/diode array detection (HPLC/DAD). Washing solvent was optimized in terms of kind and volume for removing the matrix constituents nonspecifically adsorbed on the MIP. It has been shown that the nonspecific binding ability of the sulfonylureas to the polymer largely increased along with increasing the concentration of Ca2+ ions in the water sample, whereas complexation of divalent ions with EDTA eliminated this interference completely. The stability of MIP was tested by consecutive percolation of water sample, and it was shown that the performance of the MIP did not vary even after 200 enrichment and desorption cycles. Recoveries of the five sulfonylureas extracted from 1 L of tap water and surface water samples such as river water and rainwater at a 50 ng/L spike level were not lower than 96%. The recoveries of sulfonylureas extracted from 10-g soil sample at the 50 microg/kg level were in the range of 71-139%. Depending on the particular compound, the limit of detection varied from 2 to 14 ng/L in water and from 5 to 12 microg/kg in soil samples. The MIP was also compared with a commercially available C-18 column and an immunoaffinity support with encapsulated polyclonal anti-MSM antibodies in sol-gel glass.

Acrylates↗

PubMed2002

Photocatalytic atrazine degradation by synthetic minerals, atmospheric aerosols, and soil particles.

In this work, the photocatalytic atrazine degradation by seven synthetic minerals and five environmental particle samples was examined to investigate a possible contribution of photocatalysis to the abiotic degradation of atrazine in the environment. Particle suspensions containing 500 ng/L atrazine were irradiated with a sun simulator, and the atrazine degradation was monitored by enzyme-linked immunosorbent assay (ELISA). Atrazine detection by ELISA proved to be an useful analytical tool because of low cross-reactivity of atrazine metabolites and high sensitivity with detection limits in the lower nanograms per liter range. The atrazine degradation followed first-order kinetics, and the obtained rate coefficients were compared with the rate of direct photolysis. Known photocatalysts, such as TiO2 and ZnO, showed the expected fast photocatalytic degradation (k = 27-327 x 10(-3) min(-1)) of atrazine. The degradation rates detected upon irradiation of titanium-, zinc-, or iron-containing minerals were orders of magnitudes lower (k = 0.15-0.70 x 10(-3) min(-1)) but still significantly faster than direct photolysis without particles (k = 0.10 x 10(-3) min(-1)). With environmental particle samples (soot, fly ash, sand, road dust, and volcanic ash), however, no significant photocatalytic activity was observed (k = 0.07-0.16 x 10(-3) min(-1)). The atrazine degradation rates were in the range of direct photolysis. Thus photocatalysis by aerosol or soil particles appears not to enhance abiotic atrazine degradation in the environment.

Aerosols↗

PubMed2002

Process analysis of recycled thermoplasts from consumer electronics by laser-induced plasma spectroscopy.

An experimental setup for direct elemental analysis of recycled thermoplasts from consumer electronics by laser-induced plasma spectroscopy (LIPS, or laser-induced breakdown spectroscopy, LIBS) was realized. The combination of a echelle spectrograph, featuring a high resolution with a broad spectral coverage, with multivariate methods, such as PLS, PCR, and variable subset selection via a genetic algorithm, resulted in considerable improvements in selectivity and sensitivity for this complex matrix. With a normalization to carbon as internal standard, the limits of detection were in the ppm range. A preliminary pattern recognition study points to the possibility of polymer recognition via the line-rich echelle spectra. Several experiments at an extruder within a recycling plant demonstrated successfully the capability of LIPS for different kinds of routine on-line process analysis.

Electronics↗