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In recent years, the sol–gel technique has attracted increasing interest as a unique approach to immobilize biomolecules for bioanalytical applications as well as biochemical and biophysical studies. For this purpose, crushed biomolecule-doped sol–gel glass monoliths have been widely used. In the present work, for the first time, the encapsulation of anti-diclofenac antibodies in silica nanoparticles was carried out by a combination of reverse micelle and sol–gel technique. Cyclohexane was used for the preparation of the microemulsion as organic solvent, while surfactant Igepal CO-520 was found to be the optimal stabilizer. The antibody source was a purified IgG fraction originating from a polyclonal rabbit antiserum. Tetramethyl orthosilicate (TMOS) was used as precursor. Rather uniform, monodispersed and spherical silica particles of about 70 nm diameter size were fabricated, as was demonstrated by transmission electron microscopy (TEM) and scanning electron microscopy/energy dispersive X-ray fluorescence analysis (SEM/EDX). The biological activity of the encapsulated antibodies was evaluated by incubation of the nanoparticles with a diclofenac standard solution and analysis of the filtrate and followed washing solutions by a highly sensitive enzyme-linked immunosorbent assay (ELISA), using non-doped particles as blanks. While only about 6% of the added diclofenac was nonspecifically retained by the blank, the corresponding amount of about 66% was much higher with the antibody-doped particles. An obvious advantage of this approach is the general applicability of the developed technique for a mild immobilization of different antibody species.

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.

Immunological techniques are recognized as rapid, sensitive, and inexpensive methods in environmental analytical chemistry. By screening out negative samples (no target analyte present or only detectable at levels below a set threshold limit), they can constitute a valuable tool to decrease the demand for chemical analyses by more time-consuming and highly sophisticated instrumentation. Currently, corresponding tests are available mainly in the microtiter plate and tube format. A limitation of an immunoassay as compared to common chromatographic techniques often specified by environmental analysts consists in its single-compound analysis. Because antibodies target epitopes, not the whole antigen, multiple substances may have same or similar epitopes. When this is the case, the crossreactivity of antibodies can be used for a multiresidue analysis of structurally related compounds that might be a class of pesticides, such as triazines including a number of metabolites. Increasing activities in the validation and standardization of immunochemical tests, as were initiated by federal government and regulatory agencies of several countries and well-recognized national and international organizations, will lead to more transparency and uniformity in method development and evaluation, and therefore will aid the assays in getting rid of their smack as “dubious biological tests.”

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.

Application of capillary isotachophoresis (CITP) for the analysis of water extracts of the dust samples collected in different periods in air-filtration devices in Prague car traffic tunnels and in Parisian metro station is presented. The extracts were analyzed in cationic mode with a leading electrolyte (LE) of 10 mM KOH, 25 mM acetic acid, pH 4.4, and a terminating electrolyte (TE) of 10 mM beta-alanine, adjusted to pH 4.4 with acetic acid, and in anionic mode with LE 10 mM HCl, 20 mM histidine, pH 5.8 and TE 10 mM 2-(N-morpholino)ethanesulphonic acid, pH 3.7. Extracted amounts of UV-absorbing substances, including pollen allergens and organic pollutants, the number of the found components and concentrations of some inorganic ions (e.g. Cl-, K+, Na+, Ca2+) in the dust samples were determined. It was found that the extracted amounts of anionic components and their number were much higher than those of cationic components. Significant differences have been found in the analyses of the extracts of different origin. Much more material and more components were present in the extracts of samples from the pollen-rich period than from the pollen-free period, especially in anionic CITP mode.

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.

A rapid and efficient method for the selective extraction of s-triazine herbicides in environmental samples was developed using an immunosorbent of monoclonal antiatrazine antibodies, which were encapsulated in a sol-gel glass matrix. The cross-reactivity of the antibody for analytes structurally related with atrazine enabled the simultaneous extraction of several s-triazine herbicides (atrazine, propazine, terbuthylazine, cyanazine, desethyl atrazine). After trace enrichment on the immunoextraction column, the s-triazines were desorbed by means of an acidic buffer (pH 2.5) and further extracted with ethyl acetate before being injected into the GC. Compared to liquid-liquid extraction and solid-phase extraction with a hydrophobic SDB-L support, the GC-NPD chromatograms obtained after immunoaffinity enrichment of surface water (river water) samples or soil extracts and analysis were free from matrix interferences. Nonspecific adsorption of humic acids was not observed. The method allows for the determination of the herbicides in linear ranges up to 1.5 microg/L with correlation coefficients higherthan 0.99 and relative standard deviations between 4% and 7% (n = 5). The LODs for 50-mL water samples were in the range 0.02 microg/L (atrazine, propazine) to 0.1 microg/L (desethyl atrazine) (S/N = 3). In addition to its high selectivity, the immunosorbent proved to be reusable for a significant number of preconcentration runs. However, the composition of samples may influence the lifetime of the column.

In recent decades an increased prevalence of allergic conditions has been observed in developed countries. Although lifestyles, exposure to infection, and diet are all likely important factors, many studies have also shown a strong link between industrialization and allergy. The aim of this study was to investigate which extract fractions from traffic particulate matter (TPM, collected in a tunnel in Prague) have the greatest impact on different inflammatory and immunological parameters, such as cytokine production, levels of immunoglobulin E (IgE) antibodies, and bronchial hyperresponsiveness (BHR) in mice, when the extracts are used together with birch pollen for immunization. BP2 mice were immunized with birch pollen and different fractions of TPM (fractions 1-8). They were provoked intranasally with a mixture of pollen and TPM or pollen alone before they were challenged with methacholine. The BHR was evaluated in a whole-body plethysmograph. Th2 cytokines and fibronectin concentrations were measured, and differential cell counts were performed in the bronchioalveolar lavage (BAL) fluid. Sera were collected for determination of antibody titers. The highest titers of IgE and the highest BHR were found in the positive control mice (immunized and provoked with a mixture of pollen and TPM), followed by mice immunized with pollen and fraction 2 (which contains organic acids). Fraction 2 also induced the highest number of eosinophils and increased levels of interleukin 5 (IL-5) in the BAL fluid. The highest levels of IL-5, in BAL fluid and sera, were obtained in mice immunized with fraction 6 (moderately polar compounds), a somewhat surprising result since those mice did not produce any IgE, did not have any eosinophils in their BAL, or showed almost no BHR. Our data demonstrate that fractions 2 (organic acids) and 7 (highly polar compounds) seem to contain potential adjuvants stimulating the IL-5 production, the IgE synthesis, the eosinophil recruitment, and the bronchial hyperreactivity. Further characterization at the molecular level is now necessary to be able to identify the exact nature of those potential adjuvants. This will be of help in the future to improve the quality of the urban air aerosol.

A new, rapid method for selective extraction of hydroxylated polycyclic aromatic hydrocarbons metabolites (OH-PAHs) in human urine was developed using an immunosorbent of anti-pyrene antibodies which were encapsulated in a sol-gel glass (SGG) matrix. Resulting chromatograms after immunoextraction of urine samples and HPLC analysis of the extracts were free from matrix interferences. The LODs for the determination of OH-PAHs in these difficult samples were in the low-ppt range (1-16 ng/L). In addition to its high selectivity, the immunosorbent proved to be robust and reusable. Obtained recoveries in spiked urine samples ranged from 83 to 107% for the hydroxyphenanthrene and hydroxypyrene compounds under investigation, while recovery for 3-hydroxybenzo[a]pyrene was only 45-62%. In a biomonitoring study, the SGG immunosorbent was successfully used for trace-level analysis of OH-PAHs in 20 human urine samples. Results were compared to data obtained by an independent reference analysis method and revealed good correlation between both methods.

Benzo[a]pyrenebutyric acid (B[a]PBA) has been synthesized and covalently coupled to bovine serum albumin to generate monoclonal antibodies (Mab). A competitive indirect enzyme-linked immunosorbent assay (ELISA) for polycyclic aromatic hydrocarbons (PAH) has been developed with Mab B[a]P-13. It was shown by testing with 21 parent PAH and 10 compounds carrying methyl, hydroxy, or butyric acid functions that the antibody had broad specificity. Highest affinity was found for four- to six-ring PAH. Different organic co-solvents were tested. No loss in sensitivity, compared with controls in PBS, were found with methanol, dimethyl sulfoxide, and glycerol at final concentrations of 5 to 10%. Further, an observation was made that a modification (fine-tuning) of the affinity and specificity of the antibodies was possible by changing the type of the added organic co-solvent. The high susceptibility of the ELISA with regard to inorganic ions might be an indication of a more hydrophilic binding pocket e.g. involving a pi-cation interaction. Investigation of the effect of pH revealed that for pH between 6 to 9 there was no noticeable impairment. With an LOD as low as 30 pg per well for B[a]P the sensitivity of the ELISA is sufficient for analyses of solvent extracts of many environmental samples. As an example, the determination of a PAH sum parameter, given as B[a]P-equivalents, in crude aerosol extracts by both ELISA and HPLC revealed good correlation (r2=0.717) but approximately five-fold overestimation by the immunochemical method, obviously as a result of cross-reacting analytes.

The quality of air is one of the most important factors that affect life quality and public health. Many sources of pollution are threatening this precious good, e.g., emissions from traffic and industry, but natural particles like pollen, spores, viruses or bacteria as well. To get an adequate impression of the degree of the air pollution, monitoring of all pollutants or at least of the most relevant ones (considering their amount and their harmfulness to human health) would be necessary. By determining only one class of compounds little can be concluded on the total effect of the pollutants on human health. Additionally synergistic effects of different pollutants may lead to increased derogation. One example from recent literature is the potential of polycyclic aromatic hydrocarbons (PAHs) to induce allergies (Diaz-Sanchez 1997, Bommel et al. 2000).

An enzyme-linked immunoassay (ELISA) for the determination of humic acid in natural-water samples was developed. For antisera production, humic acid was coupled to helix pomatia hemocyanin. A humic acid-horseradish peroxidase conjugate was synthesized as a tracer. The detection limit of this competitive assay was estimated to be 0.5μg/l, which means 100pg absolute. The cross-reactivities at the center point (50%-value) of several commercial humic acids and both aliphatic and aromatic acids were determined. Moreover, the structural aspects of the humic acids could be elucidated.

Complete 23S and almost complete 16S rRNA gene sequences were determined for the type strains of the validly described Enterococcus species, Melissococcus pluton and Tetragenococcus halophilus. A comprehensive set of rRNA targeted specific oligonucleotide hybridization probes was designed according to the multiple probe concept. In silico probe design and evaluation was performed using the respective tools of the ARB program package in combination with the ARB databases comprising the currently available 16S as well as 23S rRNA primary structures. The probes were optimized with respect to their application for reverse hybridization in microplate format. The target comprising 16S and 23S rDNA was amplified and labeled by PCR (polymerase chain reaction) using general primers targeting a wide spectrum of bacteria. Alternatively, amplification of two adjacent rDNA fragments of enterococci was performed by using specific primers. In vitro evaluation of the probe set was done including all Enterococcus type strains, and a selection of other representatives of the gram-positive bacteria with a low genomic DNA G+C content. The optimized probe set was used to analyze enriched drinking water samples as well as original samples from waste water treatment plants.

An enzyme-linked immunosorbent assay (ELISA) on microplates and a HPLC coupled-column switching method were compared for the determination of the nitroarene compound 1-nitropyrene in airborne particulate organic matter collected at a busy intersection over a period of 2 months. After purification of the sample extract with silica, in the multidimensional chromatographic method nitroarenes were separated on a RP18 precolumn from matrix constituents followed by on-line reduction to corresponding aminoarenes with a Pt catalyst on alumina and a further separation of 1-aminopyrene on a second RP18 column. Methanol-water (70:30, v/v) was the mobile phase used. With ELISA, a six-fold overestimation was obtained for untreated samples. After clean-up it was lowered to approximately 1.6-fold overestimation which was mainly caused by cross-reaction of 2-nitropyrene and 2-nitrofluoranthene.

To investigate the applicability of immunological methods as sensitive and cost-effective analytical screening tools a pyrene immunoassay was developed that can be used for the semiquantitative detection of PAHs in water and soil. While contaminated groundwater samples from a former gas plant site could be analyzed directly, soil and seepage water has to be extracted and require at least 1:100 dilution prior to immunochemical measurement. PAHs could be recovered from fortified reference soils as well as aged field samples with high yield using 1-hour ultrasonication with acetonitrile. Extraction efficiency was lower with agitation but was acceptable as part of an on-site field test to provide rapid, semiquantitative, and reliable test results for making environmental decisions such as identifying “hot spots”, site mapping, monitoring of remediation processes, and selecting site samples for laboratory analysis. Using classification of ELISA data an estimation of the PAH contamination in soils (n=18) was possible put up with about 5% false positive and 5% false negative results that may be caused by the heterogeneity of samples, PAH-profile, cross-reactive compounds, or unknown matrix interferences.

Groundwater contamination and PAH-removal with a special water purification facility were monitored over several months by a PAH-immunoassay as well as by HPLC. In the enzyme- linked immunosorbent assay (ELISA) a polyclonal rabbit antiserum was applied. As test format an indirect competitive microtiter plate assay was used. The water samples could be directly measured without any extraction and purification steps. With the ELISA, the limit value for the sum of PAHs of 0.2 ppb as set by the German Drinking Water Act was certainly reached. Including 114 samples no false negative (defined as a measured concentration below 0.2 ppb) but 20 (17.5%) false positive (defined as a measured concentration above 0.2 ppb) samples were found with the immunoassay. Supposing the immunochemical results to be approximate values of the total PAH concentration then these data correlate very well (r equals 0.82, n equals 114) with the sum of the 16 EPA PAHs as measured by HPLC in this study. Therefore, the ELISA could be used as a rapid screening technique to diminish the sample number for HPLC.© (1995) COPYRIGHT SPIE--The International Society for Optical Engineering. Downloading of the abstract is permitted for personal use only.

A simple and sensitive fluorescence immunoassay for the detection of aflatoxin B(1) (AFB(1), as a model compound) in food was developed using AFB(1)-bovine serum albumin conjugate (AFB(1)-BSA)-functionalized magnetic beads as immunosensing probes. The recognition elements were prepared by doping of rhodamine B (RB) fluorophore into silica nanoparticles followed by immobilization of monoclonal anti-AFB(1) antibodies on the silica shell. Based on a competitive-type immunoassay format, the assay was performed both in low-binding polypropylene 96-well microtiter plates (MTPs) and in an automated sequential injection (SI) format. Similar detection limit (LOD) of 0.2 ng mL(-1)vs. 0.1 ng mL(-1) but narrower dynamic working linear range of 0.5-7 ng mL(-1)vs. 0.5-30 ng mL(-1) was obtained toward AFB(1) standards with the flow setup compared to the MTP format. Intra-batch assay precision was substantially improved (≤5.3% vs.≤8.7%) by resorting to the SI manifold. The proposed method features unbiased identification of negative (blank) and positive samples. No significant differences at the 95% confidence level were encountered in the analysis of naturally contaminated peanut samples between the proposed immunoassay and liquid chromatography for determination of AFB(1).

A sensitive, automated micro flow-through chemiluminescence enzyme immunoassay (CLEIA) was developed for the determination of 2,4-dinitrotoluene (DNT) which employed a DNT analog covalently immobilized on a glass microchip, and nanogold particles (AuNPs) modified with HRP-labeled anti-mouse antibodies. The HRP-luminol-H2O2 chemiluminescent system (because of its high sensitivity) was chosen as the detection system. The obtained chemiluminescence (CL) intensity was inversely proportional to the DNT concentration in the sample. The working range for DNT quantification was 0.01–1 μg mL−1. Both the intra- and inter-assay coefficients of variation were less than 10%. The proposed assay had a LOD of 0.009 μg mL−1, which was 3-fold lower than a traditional HRP-labeled anti-mouse antibody-based CLEIA. The overall assay time was 13 min for each sample, enabling relatively rapid analysis.

A sensitive and reusable electrochemical immunoassay for aflatoxin B(1) (AFB(1)) in food has been developed. A multifunctional magnetic bead (MMB) was initially synthesized using magnetic CoFe(2)O(4) nanoparticle as the core and Prussian blue nanoparticle (PBNP)-doped silica as the shell, and then the prepared MMB was used as an affinity support for the immobilization of the AFB(1)-bovine serum albumin conjugate (AFB(1)-BSA). With the aid of an external magnet, the AFB(1)-BSA-conjugated MMBs were attached on the surface of an indium tin oxide (ITO) electrode. Gold nanoparticles, labeled with horseradish peroxidase (HRP)-bound anti-AFB(1) antibodies (HRP-anti-AFB(1)), were employed as detection antibodies. With a competitive immunoassay format, the concentrations of AFB(1) in samples were measured in PBS (pH 7.0) by using PBNP-doped MMBs as the mediator, HRP-anti-AFB(1) as the tracer and hydrogen peroxide (H(2)O(2)) as the enzyme substrate, and the linear range was 0.05-12 ng/mL with a detection limit of 6.0 pg/mL AFB(1) (at 3sigma). Intra- and inter-assay coefficients of variation were less than 7.5%. In addition, the content of AFB(1) in red paprika specimens has been assayed by the developed immunoassay and a commercially available enzyme-linked immunosorbent assay (ELISA) method, respectively, and consistent results were obtained. The as-prepared immunoassay provides a promising approach for the screening of organic pollutants because it is simple, rapid, highly sensitive, specific, and without the need of sample pre-concentration.

A new approach to generate uniform-sized nanoliter droplets inside a simple microfluidic chip was developed by using a circular groove to break off the liquid infused from the surrounded inlet. We characterized the droplet formation by using circular grooves with different diameters ranging from 1.2 to 2.4 mm. Monodisperse droplets with a very narrow size distribution (RSD < 0.56%, n = 70 droplets) can be reliably produced and the volume of droplets varied between 74 and 576 nl by using different circular grooves. Influences of liquid properties on droplet formation were also investigated, including surface tension, viscosity and density. Moreover, the transport of droplets and reliable coalescing of two neighboring droplets were realized by using Y-shaped guiding lanes with two different designs. Controlling the velocities of each droplet based on their different physical properties or slowing down the first droplet relying on a pinched segment of the guiding lane has been developed. These methods have been used to control the contact conditions of two reagents coalescing in a 'head-to-tail' mode.

Environmental Context.Benzene, toluene, ethylbenzene, and xylenes (BTEX) are used as solvents in paints and coatings and are constituents of petroleum products. BTEX can contaminate air, water or soil and is toxic; benzene, in particular, is a recognized human carcinogen. Most existing methods for detecting BTEX are time-consuming, complicated and very expensive for routine screening. A rapid immunoassay of BTEX is presented that greatly simplifies environmental monitoring of water contamination. Abstract.For the rapid screening of BTEX (benzene, toluene, ethylbenzene, xylenes), a fluorescence polarization immunoassay (FPIA) was developed using the fluorescence polarization analyzer Abbott TDx. Several fluorescence-labelled tracers were synthesized by binding ethylenediamine fluorescein thiocarbamyl (EDF) to various substituted phenylcarboxylic acids. The binding of 27 tracers with two antisera that can be considered as class-specific for BTEX was investigated to select optimal tracer–antibody pairs. Significant differences were found in binding, titer, sensitivity, and assay kinetics. A best pair of anti-BTEX antiserum and EDF-labelled p-tolylacetic acid tracer was selected for FPIA. To simplify the method, an immunocomplex single reagent was prepared to detect BTEX by a one-step FPIA. One-step FPIA is a rapid homogeneous type of immunoassay that has only one pipetting step, does not need separation of free and bound analyte and can be performed at room temperature. The within-run coefficient of variation was ranged between 3.4% and 5.7%. Furthermore, if the measurement can be done at constant temperature, standards for every assay run are unnecessary. Cross-reactivity studies of petroleum compounds and a BTEX mixture indicated that p-xylene was most reactive with a limit of detection (LOD) of 0.22 µg mL−1 in 50 µL of sample. The LOD for toluene and benzene was 2.1 and 11 µg mL−1 respectively. The immunocomplex single reagent has proven to be significantly more stable than the corresponding solutions of antibody and tracer.

More than twenty enzymes (oxidoreductases, hydrolases, lyases, transferases) from crude bacterial extracts or commercial preparations have been purified by charge-controlled hydrophobic chromatography on 10-car☐ydecyl-Sepharose. The enzymes were adsorbed on columns in the presence of high concentrations of structure-forming anions (phosphate, sulphate, citrate) and were eluted by decreasing the concentration of the salts. As a rule crystalline enzyme preparations were obtained by dialysis of eluates against ammonium sulphate solutions. These results suggest the existence of a true affinity principle based on the ability of structure-forming anions to modify the enzyme conformation. The only supposition is the immobilization of a ligand with a specific combination of hydrophobic and ionic properties in that manner, that the columns are not able to bind enzymes in the presence of dilute buffer solutions.

Synthesis and X-ray data of 3-hydroxy-N-methyl-hasubanan A new simple way to synthesis 3-hydroxy-N-methyl-hasubanan (1c) is described and a single crystal X-ray structure analysis is presented.

A direct competitive ELISA for the detection of the herbicide 2,4-D (2,4-dichlorophenoxyacetic acid) has been developed. The optimization, evaluation and characterization of the ELISA, with two different antisera, has been carried out. The detection limit of 2,4-D with antiserum 7/89 was 20 ng/l and a center point value of 0.6 μg/l 2,4-D. Antiserum 16/88 showed a detection limit of 0.3 μg/l with a center point value of 4 μg/l. The cross reactivity both at the center point and at the detection limit for five structural related phenoxycarboxylic acid herbicides has been determined. The influence of increasing concentrations of humic acid, solvents and surfactants on the ELISA has been investigated, leading to a deterioration of detection limits and center points.

Summary Among the modern molecular techniques for the identification of microorganisms the most straightforward way is through direct hybridization with rRNA/rDNA targeted probes. In this study, the optimization of the experimental procedures for the reverse hybridization technique in 96-well microplates is described using both synthetic model oligonucleotides (18 b) and amplified DNA (app. 4500 bp). Three different types of plates were compared (Maxi Sorp, NucleoLink, CovaLink). Plates made from non-chemically modified polystyrene which are conventionally used in immunoassays (MaxiSorp) proved to be an economic alternative for plates offering chemically modified tailor-made surfaces. Phosphorylation of the oligonucleotide probe was not necessary for successful immobilization whereas with 5′-terminal hexa-deoxyadenosine tailed capture oligonucleotides an enhanced sensitivity of the assay was observed. Variation of the stringency by adjusting different concentrations of formamide during the washing step ensures high probe specificity and therefore allows reliable identification of the microorganisms. The assay can be performed in less than 4 hours using pre-coated plates which can be stored for several weeks. After dissociation of the target DNA/capture probe duplex with an alkaline denaturing solution rehybridization is possible.

A polarization fluoroimmunoassay (PFIA) based on previously raised rabbit polyclonal antibodies was developed for detection and quantification of the sulfonylurea herbicide metsulfuron-methyl (MSM) in surface water samples. A 'restricted' hapten, consisting of only phenylsulfonamide moiety of MSM molecule was used to synthesize a fluorescein-labelled derivative of MSM (tracer). The limited assay cross-reactivity was observed towards most of sulfonylurea herbicides, with exception of primisulfuron-methyl and chlorimuron-ethyl. The optimized MSM PFIA had a dynamic range from 30 to 1000 w g r -1 with an IC 50 value of 123 w g r -1 and a lower detection limit of 5 w g r -1 . Intra- and inter-assay precision over the of MSM calibration curve dynamic range were 0.9 and 7.1%, respectively. Influence of several organic solvents on MSM PFIA performance was investigated, and the assay was found to tolerate up to 10% v/v of methanol, DMSO and acetone. Application of MSM PFIA technique to the analysis of surface wate...

Polycyclic aromatic hydrocarbons (PAHs) represent health and environmental concerns. The present study was designed to develop a highly sensitive and reliable immunochemically-based non-instrumental field assay to monitor the presence of benzo[a]pyrene (BAP) in aqueous samples at the ppt-level using visible test evaluation. The corresponding gel-based immunoassay was set up to combine preconcentration and detection of the target analyte using anti-PAH antibodies and horseradish-BAP tracer conjugate in one single cartridge. Water sample preparation, such as extraction, centrifugation, or filtering was found to be unnecessary. No interference by higher water-soluble PAHs at 4000-fold excess compared to BAP was observed. The assay was configured as a qualitative test (positive/negative) at the cut-off level of 5 ng L(-1), however, this level can be adapted to the required analyte concentration by rather simple adjustment of the anti-PAH antibody concentration of the test zone. Test validation was performed with real samples such as surface water, melted snow, and tap water using high performance liquid chromatography with fluorescent detection (HPLC-FLD) with solid phase extraction for validation.

Antisera to 2,4-dichlorophenoxyacetic acid (2,4-D), a widely used herbicide, have been obtained from rabbits following immunization with various 2,4-D-protein conjugates. Employing [125I] 2,4-D-tyramine as the radioligand for the antisera, very poor assay sensitivity was achieved because of a much higher affinity of the antibodies to the tracer. When using [6-3H] 2,4-D(specific radioactivity 465 GBq/mmol) a sensitive and specific radioimmunoassay (RIA) for 2,4-D could be developed, which allows determination directly in water, plasma and urine samples. Levels as low as approximately 100 pg (450 femtomoles) of 2,4-D can be detected. The antiserum is fairly specific for 2,4-D. Other related phenoxycarboxylic acids and dichlorophenol showed a cross-reactivity smaller than 10%. After a single administration of 2,4-D (0.91 mg/100 g body weight, orally) to rats, plasma and urine levels were determined at different times. Results correspond to those found in the literature, thus indicating the utility of the RIA. Further applications and limitations are discussed.