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ZusammenfassungEchtzeitmessungen auf der Internationalen Raumstation (ISS) konnten belegen, dass Zellen prinzipiell in der Lage sind, sich ultraschnell an veränderte Schwerkraftverhältnisse zu adaptieren. Im Experiment TRIPLE LUX A wurde der Einfluss von Gravitationskräften zwischen 1 und 0 g auf die Oxidative-Burst-Reaktion von NR8383-Alveolarmakrophagen untersucht. Erstmals konnte durch Echtzeit- und On-orbit-Messungen auf der ISS eine schwerkraftabhängige Reaktion in Säugerzellen direkt bewiesen und verfolgt werden. Durch den Messaufbau gelang ebenso der erstmalige Nachweis einer ultraschnellen und nach 42 Sekunden erfolgten vollständigen Anpassung einer zellulären Reaktion an die Schwerelosigkeit.

ZusammenfassungAm Ende sah es wie Routine aus: Am 14. April 2015 um 20:10:41 UTC startete vom Cape Canaveral Launch Complex SLC-40 eine Space X Falcon 9 Rakete mit dem Dragon-Raumschiff (Abb. 1) zur sechsten „Cargo Resupply Mission“ (Space X CRS-6) zur Internationalen Raumstation ISS. Mit an Bord: Die komplette Experimenthardware, tiefgefrorene Zellen und Reagenzien des DLR-Experiments TRIPLE LUX A. Im Experiment sollte der Einfluss von Gravitationskräften zwischen 1 und 0 g auf die Oxidative-Burst-Reaktion von Makrophagenzellen untersucht werden, erstmals durch Echtzeit- und On-orbit-Messungen auf der ISS im BIOLAB des europäischen COLUMBUS-Moduls 1.

Despite the observed severe effects of microgravity on mammalian cells, many astronauts have completed long term stays in space without suffering from severe health problems. This raises questions about the cellular capacity for adaptation to a new gravitational environment. The International Space Station (ISS) experiment TRIPLE LUX A, performed in the BIOLAB laboratory of the ISS COLUMBUS module, allowed for the first time the direct measurement of a cellular function in real time and on orbit. We measured the oxidative burst reaction in mammalian macrophages (NR8383 rat alveolar macrophages) exposed to a centrifuge regime of internal 0 g and 1 g controls and step-wise increase or decrease of the gravitational force in four independent experiments. Surprisingly, we found that these macrophages adapted to microgravity in an ultra-fast manner within seconds, after an immediate inhibitory effect on the oxidative burst reaction. For the first time, we provided direct evidence of cellular sensitivity to gravity, through real-time on orbit measurements and by using an experimental system, in which all factors except gravity were constant. The surprisingly ultra-fast adaptation to microgravity indicates that mammalian macrophages are equipped with a highly efficient adaptation potential to a low gravity environment. This opens new avenues for the exploration of adaptation of mammalian cells to gravitational changes.

The development of a monoclonal antibody enables a specific and sensitive detection of a potentially allergenic epitope from proglycinin in soy and constitutes an appropriate tool for analytical purposes in medical diagnostics and food labeling. Proglycinin was utilized as immunogen. The most favorable antibody G17E detected in a hybridoma cell culture screening was mass produced, purified, and used for immunoaffinity purification of proglycinin. In a heterogeneous, non-competitive ELISA format, a 50% ELISA inhibition point of 13 µg/L for purified proglycinin was determined. Cross-reactivity with homologous epitopes could be excluded. Specificity of G17E was determined by epitope mapping.

The analysis of gravisensing in fungi has not yet proceeded as far as in other systems, such as sensory cells for maintenance of equilibrium in crustaceans and vertebrates, Chara rhizoids or plant roots with regard to positive gravitropism. However, considerable progress has recently been made with several fungal systems in the field of graviresponses. This particularly holds true for the molecular basis of primary events. In this review three systems of increasing complexity are considered: the slime molds Physarum and Dictyostelium, which exhibit gravitaxis, the negative gravitropic sporangium of the zygomycete Phycomyces as a single cell structure with the fastest graviresponses known so far, and the fruit bodies of the basidiomycetes Coprinus and Flammulina as highly complex structures with the ability to generate positional information and cell signalling in the context of negative gravitropism.

Mammalian soluble epoxide hydrolase (sEH) represents a highly promising new target for drug development. Chemical inhibition of this enzyme in animal models was shown to treat hypertension and vascular inflammation as well as related syndromes. Existing sEH inhibitors are relatively potent and specific. However, the low solubility and relatively fast metabolism of described sEH inhibitors make them less than therapeutically efficient, stating the need for novel inhibitor structures. Therefore, a series of alpha-cyanoester and alpha-cyanocarbonate epoxides were evaluated as potential human sEH (HsEH) substrates for the high-throughput screen (HTS) of compound libraries. (3-Phenyl-oxiranyl)-acetic acid cyano-(6-methoxy-naphthalen-2-yl)-methyl ester (PHOME), which displayed the highest aqueous stability and solubility, was selected for the development of an HTS assay with long incubation times at room temperature. Concentrations of HsEH and PHOME were optimized to ensure assay sensitivity, reliability, and reproducibility. Assay validation, which employed these optimized concentrations, resulted in good accuracy (60-100%) and high precision (7% relative standard deviation). In addition, an overall Z' value of 0.7 proved the system's robustness and potential for HTS. The developed assay system will be a valuable tool to discover new structures for the therapeutic inhibition of sEH to treat various cardiovascular diseases.

It is now known that many organochlorines (OCs) act as endocrine disruptors, causing harmful effects on wildlife and humans. Several field and laboratory animal studies have reported that OCs cause adverse effects on thyroid hormone status. However, data regarding their effects on thyroid hormone status in humans are inconclusive. Because a developing fetus is especially sensitive to hormonal disruption by exposure to OCs, the adverse health effects on infants are of concern. The present study aimed to investigate the association between OC levels in maternal and cord serum, and the association between OC and thyroid hormone levels in cord serum. The study was performed with 39 mother–infant pairs from Mae Rim District of Chiang Mai Province, northern Thailand, who had normal delivery and full term gestation. Maternal blood was collected for measuring OCs and total lipids. Umbilical cord blood was collected for measuring OCs, total lipids, and thyroid hormones, including total thyroxine (TT4), free thyroxine (FT4), and thyroid stimulating hormone (TSH). 1,1-dichloro-2,2-di(4-chlorophenyl)ethylene (p,p′-DDE) had the highest level in all serum samples with a geometric mean of 1191 ng/g lipids in maternal serum and 742 ng/g lipids in cord serum. The second highest level was that for 1,1,1-trichloro-2,2-di(4-chlorophenyl)ethane (p,p′-DDT), followed by 1,1-dichloro-2,2-di(4-chlorophenyl)ethane (p,p′-DDD). Levels of p,p′-DDE, p,p′-DDT, p,p′-DDD, and dieldrin in maternal serum were positively associated with levels in cord serum (r=0.86, 0.77, 0.66, and 0.60, respectively; P

Gene expression patterns are efficient tools to elucidate responses to hormones as well as endocrine disruptors at the molecular level. Male zebrafish (Danio rerio) were exposed to 17β-estradiol (E...

Inhibition of the mammalian soluble epoxide hydrolase (sEH) is a promising new therapy in the treatment of disorders resulting from hypertension and vascular inflammation. A spectrophotometric assay (4-nitrophenyl-trans-2,3-epoxy-3-phenylpropyl carbonate, NEPC) is currently used to screen libraries of chemicals; however this assay lacks the required sensitivity to differentiate the most potent inhibitors. A series of fluorescent alpha-cyanoester and alpha-cyanocarbonate epoxides that produce a strong fluorescent signal on epoxide hydrolysis by both human and murine sEH were designed as potential substrates for an in vitro inhibition assay. The murine enzyme showed a broad range of specificities, whereas the human enzyme showed the highest specificity for cyano(6-methoxy-naphthalen-2-yl)methyl trans-[(3-phenyloxiran-2-yl)methyl] carbonate. An in vitro inhibition assay was developed using this substrate and recombinant enzyme. The utility of the fluorescent assay was confirmed by determining the IC(50) values for a series of known inhibitors. The new IC(50) values were compared with those determined by spectrophotometric NEPC and radioactive tDPPO assays. The fluorescent assay ranked these inhibitors on the basis of IC(50) values, whereas the NEPC assay did not. The ranking of inhibitor potency generally agreed with that determined using the tDPPO assay. These results show that the fluorescence-based assay is a valuable tool in the development of sEH inhibitors by revealing structure-activity relationships that previously were seen only by using the costly and labor-intensive radioactive tDPPO assay.

An immunosensor based on surface plasmon resonance (SPR) was used for the detection of a low-molecular-weight analyte, domoic acid (DA) (MW 310 g/mol), using an inhibition assay. A novel surface functionalization method was developed to chemically immobilize DA molecules onto a gold-coated SPR chip functionalized with mixed oligo(ethylene glycol) (OEG) self-assembled monolayers (SAMs), where the long-chain HS(CH2)11(C2H5O)6NH2 thiol served as a functional site to react with carboxyl groups on DA while the short-chain HS(CH2)11(C2H5O)4OH thiol served as a protein non-fouling background. Control experiments showed that there was no detectable non-specific protein adsorption on the sensor surface immobilized with DA. The binding of monoclonal anti-DA antibodies onto the sensor surface was measured using SPR and the concentration of the antibody used in the inhibition assay was optimized to provide the desired detection limit and operating range. A lowest detection limit of 0.1 ng/ml DA was established using the inhibition assay. In addition, the regeneration and storage of sensor chips were studied. The detection curve obtained from a chip which was stored for 1 week and regenerated for each measurement is almost the same as that from freshly prepared chips.

Molecular techniques have become increasingly important for the identification of arbuscular mycorrhizal fungi (AMF). In this work Acaulospora longula and Glomus mosseae have been detected in plant roots from pastures using specific nucleotide primers for the two species. Part of the 5' end of the large subunit of the ribosomal RNA gene was amplified by nested PCR and sequenced. The distribution of the fungi within three different plant species, Plantago lanceolata, Trifolium repens, and Holcus lanatus, and two different types of grassland, have been studied. Neither the fungi nor the plants showed specific preference for their symbiotic partnership.

Juvenile hormone esterases (JHEs) from six insects belonging to three orders (Lepidoptera, Coleoptera, and Diptera) were compared in terms of their deduced amino acid sequence and biochemical properties. The four lepidopteran JHEs showed from 52% to 59% identity to each other and about 30% identity to the coleopteran and dipteran JHEs. The JHE of Manduca sexta was remarkably resistant to the addition of organic co-solvents and detergent; in some cases, it demonstrated significant activation of activity. Trifluoromethylketone (TFK) inhibitors with chain lengths of 8, 10 or 12 carbons were highly effective against both lepidopteran and coleopteran JHEs. The coleopteran JHE remained sensitive to TFK inhibitors with a chain length of 6 carbons, whereas the lepidopteran JHEs were significantly less sensitive. When the chain was altered to a phenethyl moiety, the coleopteran JHE remained moderately sensitive, while the lepidopteran JHEs were much less sensitive. The lepidopteran and coleopteran JHEs did not show dramatic differences in specificity to alpha-naphthyl and rho-nitrophenyl substrates. However, as the chain length of the alpha-naphthyl substrates increased from propionate to caprylate, there was a trend towards reduced activity. The JHE of M. sexta was crystallized and the properties of the crystal suggest a high-resolution structure will follow.

Biomonitoring uses biological responses to assess environmental changes, which are frequently due to anthropogenic pollution. Biomonitoring can be carried out at different levels of complexity: population, species or suborganismic responses may be used as indicators for exposure to pollutants. In the case of tests with whole organisms, their quality is often impaired by the heterogeneity of living organisms, slow responses and the fact that little information can be provided on the pollutants. Although suborganismic tests can provide more specific and fast responses, structure and concentration of pollutants are only revealed by chemical analysis. A novel approach to biomonitoring is introduced, which applies tight coupling of bioassays and chemical analysis and is defined as bioresponse‐linked instrumental analysis. It combines biomolecular recognition, initiating a biological effect and chemical analysis.

The current work describes the raising of polyclonal antibodies (pAbs) against domoic acid (DA), an algal toxin produced by the diatom Pseudonitzschia pungens. They were screened for sensitivity and selectivity using a competitive enzyme-linked immunosorbent assay (ELISA). The antiserum produced against a keyhole limpet haemocyanin (DA-KLH) conjugate displayed a high affinity for free DA. The optimized horseradish peroxidase (HRP) ELISA had a detection limit of 0.6 ng mL−1 (ppb) and a working range of 0.8–300 ppb DA applying a streptavidin-biotin amplification system (ABC system). Furthermore this antiserum did not cross-react with similar chemical structures and algal toxins such as kainic acid, aspartic acid, glutamic acid, geranic acid and 2-methyl-3-butenoic acid. When the ELISA was compared using an alkaline phosphatase (AP) label we found the system to behave in a similar manner to the optimized HRP system but the linear range was smaller in the high DA concentration range. These pAbs were ...

Monoclonal antibodies (mabs) are of great interest according to their homologous abilities, high specificity, and unlimited possibility of production. Mabs against different substances have been developed. They are used in many fields of application (e.g., medicine and pesticide analysis). Immunoassays performed with mabs have the advantages of easy handling and high sensitivity. The goal of this study was to develop mabs against 2,4,6-trichloroanisole (TCA). TCA has been identified as the most potent contaminant in wine causing a mouldy taste. Several mice have been immunized with two different TCA haptens. Spleen cells have been gained and prepared following the technique developed by Kohler and Milstein (Kohler, G.; Milstein, C. Continuous cultures of fused cells secreting antibody of predefined specificity. Nature 1975, 256, 495–497). After a screening period with a competitive immunoassay two cells lines have been detected that produce TCA-specific antibodies. Both different antibodies have ...

Biomonitoring is currently performed at two levels, assessing exposure to pollutants and effects monitoring by bioassays. As an example for the first approach, vitellogenin (VTG) in male fish of Abramis brama as an endpoint for estrogen exposure is discussed. However, similar changes of VTG or VTG-like proteins in the hemolymph of mussels could not be detected. Enzyme-linked receptor assays for monitoring estrogenic effects at the molecular level serve as an example for the second category. Applications of the enzyme-linked receptor assay (ELRA) developed in our laboratory are presented. Detection limits of 0.02 μg/l 17β-estradiol were recently achieved with the chemiluminescent format. Although effect monitoring provides information in terms of toxicity equivalents, it is not possible to relate the signals to specific pollutants and their concentrations. For this purpose, chemical analysis is required. New approaches are reported for the direct coupling of bioassays and chemical analysis. This concept is defined as bioresponse-linked instrumental analysis. It combines biomolecular recognition, initiating a biological effect, and chemical analysis. In addition to the classical bioanalytical approaches, new strategies in genomics and proteomics have been developed. This may lead to multimarker approaches opening this area to environmental analytics.

The evolutionary optimization of antibody binding propertiesin vitroopens new perspectives for immunochemistry, since the affinity and selectivity of a given antibody molecule can be tailored to meet the requirements of the envisaged analytical application. An efficient strategy for molecular antibody evolution is described that combines randomized point mutations and sequential recombination of variable antibody gene repertoires employing a group-selective library. This strategy enabled significant improvements in the binding of the model analyte atrazine that were monitored by both, kinetic measurements by the optical sensor BIAcore 2000TMand immunochemical key data obtained by enzyme-linked immunosorbent assay (ELISA). The KDof the template antibody IPR-7 was improved by a factor of 17 from 1.27×10–8M to 7.46×10–10M for the optimized variant IPR-83. The enhanced KDis well in line with the 15 fold lowered IC50of the atrazine ELISA, which was shifted from 13.6 µg/l for IPR-7 to 0.9 µg/l for IPR-83. Once the analytical properties of antibody fragments are optimized, antibody functionality can be tailored for specific technical demands, e.g. the directed immobilization on microchip surfaces. As an example, the variable region encoding genes of the scFv variants were subcloned into the Fabfragment expression vector pASK99, in order to reconstitute the antigen binding site of native antibody molecules. The expressed Fabfragments provide a C-terminal affinity tag for functionalized sensor surfaces. Again, the evaluation by ELISA as well as by BIAcore revealed a consistent ratio of analyte binding enhancement for the engineered Fabfragments.

A highly sensitive enzyme immunoassay is described for the detection of atrazine residues in water. Atrazine derivative was conjugated to Bovine Serum Albumin (BSA) to obtain an immunizing antigen and to Horseradish Peroxidase enzyme (POD) to obtain a marker for immunoassay. The formation of these conjugations was confirmed by UV spectroscopy as well as by gel-electrophoresis. Polyclonal antibodies were raised in rabbits by immunization with an atrazine-BSA conjugate containing 29 atrazine residues per BSA molecule. An ELISA on microtitration plates was optimized with peroxidase-atrazine conjugate. The middle of the test (50% B/Bo) was found to be at 90 ng/l, which is well below the maximum concentration permitted by the EC guidelines for drinking water. Detection limits for atrazine of about 1 ng/l could be reached. The assay did not require concentration or cleanup steps for drinking or ground water samples. Validation experiments showed good accuracy and precision. No cross-reactivities were shown by other s-triazines like terbutryn, ametryn, terbuthylazine, des-isopropylatrazine, and de-ethylatrazine except hydroxyatrazine. The latter was present at very low levels that can be calibrated/standardized before analysis or it may be considered as leftover residues of atrazine. Based on these results, it is suggested that this test can be applied to obtain fairly accurate results for atrazine concentration in water samples from different sources.

Monoclonal antibodies were developed against domoic acid (DA), an algal toxin produced by the diatom Pseudonitzschia pungens. Three murine monoclonal antibodies, 4D1D, 9B10D, and 7D6C, were obtained from hybridoma clones. They were derived by fusion of mouse myeloma cells with mouse immune spleen cells derived from animals immunised with domoic acid-KLH conjugate. The hybridoma cell line 4D1D was adjusted to serum free medium. It produced monoclonal antibodies of high selectivity and sensitivity. Mass production was carried out with two different fermenter systems. Monoclonal antibody levels up to 0.7 mg/ml were obtained. No cross-reactivities against similar chemical structures and algal toxins such as kainic acid, aspartic acid, glutamic acid, geranic acid and 2-methyl-3-butenoic acid could be detected. A detection limit of 4 µg/l DA with a test midpoint (IC50) of 10 µg/l DA was achieved with a competitive enzyme immunoassay applying a streptavidin-biotin amplification system (ABC system). More...

A system was constructed for the production of alkaline phosphatase (aP)-labeled antibody single-chain Fv (scFv) fragments in Escherichia coli. The expression vector pASK75 was modified by sequentially inserting the E. coli aP coding region and the scFv cloning cassette. Engineering the cloning sites SfiI and NotI located at the 5' and 3' end of the scFv gene provides an easy means to insert scFv fragments. These cloning sites are widely used in recombinant antibody technology and, thus, enable the one-step cloning of scFv fragments derived from corresponding antibody phage libraries into the expression vector. An expressed herbicide-specific scFv aP fusion protein retained both, analyte binding and enzymatic activity, as determined by ELISA. Therefore, this system permits the production of scFv-aP conjugates in E. coli, which can replace conventionally prepared aP-labeled antibodies in immunoassays. These fusion proteins are designed to accelerate the immunochemical detection of analytes, since the assay duration is essentially reduced by omitting the use of enzyme labeled secondary antibodies.

Fab antibody fragments were constructed by subcloning single chain Fv variable regions from the phagemid vector pCANTAB 5E into the expression vector pASK99. The vector was designed for bacterial secretion of Fab fragments and bears coding sequences for murine constant domains including the Strep-tag II at the carboxyl-terminal end of the constant heavy chain domain. The cloning procedure was carried out with the scFv antibodies IPR-7, IPR-53 and IPR-23. The second and third clone originated from the molecular evolution of the s-triazine selective antibody IPR-7. The Fab fragments were expressed under the transcriptional control of the tetA promoter system. Large-scale production benefits from the anhydrotetracycline-inducible system because of the lower costs for the inducer compared to IPTG and the tightly regulated expression of the recombinant antibody fragments. The Strep-tag purification technology facilitates the isolation of Fab fragments from the E. coli periplasm. The characteristics of functionally expressed Fab fragments were determined by employing a BIAcore 2000 system. The KD of the Fab variant IPR-23 (K(D)= 1.1 2 x 10(-9) M) optimized by molecular evolution was improved by a factor of 24 compared to the Fab IPR-7 (K(D) = 2.73 x 10(-8) M), which was derived from the template scFv antibody IPR-7. The affinity alteration was also reflected in the 22-fold reduction of the IC50 values of the variants Fab IPR-7 (IC50 = 60.5 microg/L) and Fab IPR-23 (IC50=2.7 microg/L) in the corresponding atrazine ELISA.

The application of immunochemical methods for the investigation of non-extractable (bound) residues is reviewed. Non-extractable residues may be presented to antibodies as antigenic determinants, which are exposed for instance in plant tissue and humic substances. Fluorescent probes as well as enzyme markers have been applied for the detection of bound residues. The application of antibodies labeled with fluorescein isothiocyanate (FITC) and phycoerythrin revealed the presence of atrazine in cryosections of atrazine-treated corn leaves and water plants. Atrazine could be localized by antibodies coupled to fluorescent markers in soil from corn fields but not in atrazine-free soil. Quantitative results were obtained by the application of enzyme immunoassays to the investigation of triazine and 2,4,6-trinitrotoluene (TNT) residues, bound to soil humic acids. Finally, the use of antibodies with different recognition patterns provides information on the ligation of non-extractable residues to the matrix.

Monoclonal antibodies were developed against vitellogenin (vtg) of rainbow trout. This protein is used as a biochemical response to the exposure with estrogenic compounds. Several mice were immunized with purified vtg and the spleen cells of these mice were fused with myeloma cells. The resulting hybridoma cells were screened with an enzyme immunoassay for the production of specific anti-vtg antibodies. Twelve positive cell lines were detected. The hybridoma cell line B8D8 was adjusted to serum free medium; it produced monoclonal antibodies with a high selectivity and sensitivity. A detection limit of 5 microg/l vtg was achieved with a competitive enzyme immunoassay applying a preincubation step and a streptavidin-biotin amplification system (ABC system). Cross-reactivity with vtg of other species was detected with roach (Rutilus rutilus), flounder (Platichthys flesus) and dab (Limanda limanda). The assay was applied to rainbow trout (Oncorhynchus mykiss), which were exposed for six months to defined concentrations of effluents (10%, 20%, 30% and 40%) and surface water. Increased vtg levels in male fish are correlated with the increasing levels of effluent. The effluent levels in the exposure experiments are relevant for effluent loadings of the Berlin waterways during the seasons of the year.

Immunochemical analysis relies on the selective binding of antibodies to defined targets such as environmental compounds. Commercial applications in the environmental field are still restricted to a limited number of immunoassays and a few immunochromatographic applications. The main barrier to a broader exploitation is seen in the generation of a sufficient number of antibodies within an acceptable time period. Recombinant technologies are expected to eventually replace the circumstantial approach to obtain new Abs by new immunizations. Examples for recombinant singlechain fragments (scFv) and antigen binding fragments (Fab) directed against herbicidal s-triazines are given. If antibodies are replaced by receptors and other functional ligands, biological effects monitoring becomes available. As an example an enzyme-linked receptor assay (ELRA) for endocrine disruptors providing estrogen equivalents is presented. Finally, the concept of bioresponse-linked instrumental analysis is introduced for t...

A monoclonal antibody (K3C6) was developed against Hg(II) and applied in different enzyme immunoassay (EIA) formats to determine the test system with the highest sensitivity. A detection limit of 1. 0 microg/L Hg(II) could be achieved with a competitive format in contrast to a detection limit of 2.1 microg/L Hg(II) with a noncompetitive EIA. A competitive displacement EIA yielded the best detection limit of 0.4 microg/L Hg(II) and was well suited to measuring real samples. For this purpose different water samples were diluted at least 1:10 to avoid matrix effects and subsequently spiked with 1 microg/L HgCl(2). Recovery of the spiked samples was between 80 and 120%.

Hormonell wirksame Substanzen in der Umwelt sorgen in den letzten Jahren immer wieder fur Schlagzeilen. Die grose strukturelle Vielfalt potenzieller Wirkstoffe erschwert allerdings deren Nachweis mit konventionellen analytischen Verfahren. Wirkungsbezogene Analysesysteme auf der Basis biomolekularer Erkennungsmechanismen bieten neue Moglichkeiten.

In the analysis of soil samples with immunochemical methods, the interpretation of tracer signals derived from marker enzymes such as peroxidase may be limited by the presence of endogenous enzyme activity. Therefore various inhibitors of peroxidase were tested for their ability to remove endogenous enzyme activity. Hydrogen peroxide, sodium azide and phenylhydrazine effectively inhibited a commercial peroxidase, while only phenylhydrazine proved to be a successful inhibitor of peroxidase in soil samples. An optimized enzyme immunoassay was then used for the investigation of soil samples for non-extractable atrazine residues. Only soil samples where atrazine had been applied during preceding years yielded positive signals in contrast to a control soil that came from a barn with no atrazine applied. Hemmung der endogenen Peroxidase in Bodenproben. Anwendung fur den Nachweis von nicht-extrahierbaren (gebundenen) Pestizidruckstanden im Enzymimmunoassay mit Peroxidase-Enzymtracer Bei der Analyse von Bodenproben mit immunchemischen Methoden kann die Auswertung des Farbsignals, produziert z.B. durch einen Peroxidase-Enzymtracer, durch endogene Enzymaktivitat des Bodens beeintrachtigt werden. Daher wurden verschiedene Peroxidase-Inhibitoren auf ihre Eignung zur Reduzierung der endogenen Peroxidaseaktivitat in Bodenproben getestet. Wasserstoffperoxid, Natriumazid und Phenylhydrazin hemmten erfolgreich eine kommerzielle Peroxidase. Allerdings konnte nur nach Anwendung von Phenylhydrazin eine ausreichende Hemmung der endogenen Peroxidase in Bodenproben festgestellt werden. Diese Methodik wurde in einem optimierten Enzymimmunoassay eingesetzt, um Bodenproben auf ihren Gehalt an nicht-extrahierbaren Atrazinruckstanden zu untersuchen. Proben von Boden, bei denen in den Jahren vor der Probennahme Atrazinanwendungen erfolgten, zeigten positive Signale, wahrend ein Kontrollboden, der unter einer Scheune lagerte, als frei von gebundenen Ruckstanden eingestuft wurde.

The single cell gel electrophoresis (SCGE) assay, or comet assay, is a highly sensitive method to measure DNA damage. The principle of the assay is based on the conversion of single-strand breaks and alkali-labile sites to double-strand breaks via alkaline lysis. The cells are subjected to electrophoresis, stained, and microscopically evaluated. Increasing damage leads to increasing migration of DNA from the nuclei. Experiments were conducted with Sordaria macrospora grown for 3 d directly on agarose minigels in order to examine the utility of the SCGE assay for the detection of effects caused by a variety of DNA-damaging agents. When the mycelia were exposed to a variety of genotoxic conditions and analyzed by SCGE, DNA strand breaks were detected by an increase in the DNA migration distance from the nucleus. The results show that this assay allows for a fast and sensitive detection of a wide spectrum of DNA-damaging effects.

The wide variety of known and unknown endocrine disruptors demands highly efficient methods for detecting the relevant endocrine disrupting pollutants. Bioeffects-related analysis is expected to solve this problem, combining biomolecular recognition processes with chemical analysis, which therefore reduces costs of instrumental chemical analysis to relevant samples. The hyphenation of both steps provides information on potential bioeffects, structure and concentration of the substances of interest. In this work the hyphenation of an enzyme-linked receptor assay (ELRA) with liquid chromatography-tandem mass spectrometry (LC-MSMS) for the analysis of estrogens and xenoestrogens is proposed. Both test systems have been validated with standards and spiked real water samples. The ELRA achieved a detection limit of 0.1 μg/L for 17β-estradiol (without preconcentration). With the LC-MSMS method, steroids could be detected down to 1 ng/L (with preconcentration). Two concepts for a tighter hyphenation of biomolecular interaction and chemical analysis are discussed: the on-line coupling of receptor-affinity chromatography (RAC) to LC-MSMS and the direct measurement of receptor-ligand complexes by LC-MSMS.

Enzyme-linked receptor assays (ELRAs) represent a bioeffects-related approach to the analysis of xenoendocrines. The receptor assay employs the same principles as competitive immunoassays based on ligand–protein interactions. However, receptor binding implicates a biological effect, either agonistic or antagonistic. In this paper, the human estrogen receptor α was used for binding estrogenic substances in environmental samples. A BIAcore biosensor was applied for receptor characterisation and assay development. The ELRA was calibrated using different estrogens and xenoestrogens including o , p ′-isomers of DDT and its metabolites. It yielded a detection limit up to 0.1 μg l −1 for 17β-estradiol. A close correlation was obtained when ELRA cross-reactivity data of different estrogenic and non-estrogenic substances were compared with data published for radioreceptor assays. Real-water samples were spiked with 17β-estradiol and measured with the ELRA. Recoveries between 90% and 100% and no significant matrix effects were observed. The ELRA can be used for cost-effective and high throughput screening of estrogens and xenoestrogens in environmental samples.

The increasing preponderance of females in fish populations has become a matter of concern not only for scientists but also for environmental authorities. For example, in the extensive waterways in Berlin, Germany, 70% of the fish population is female. A twofold question should be asked: is the biomarker vitellogenin (vitellogenin-synthesis assay) an appropriate tool for determining endocrine effects (European Commission 1996) and of chemical pollutants in effluents (Castillo et al. 1997), such as estradiols, phthalates, alkyl-phenols, and alkyl-ethoxylates, influence sex differentiation in fish?

Monoclonal antibodies (mabs) were produced against mercury (II) and an enzyme immunoassay was developed for the detection of mercury (II) in water. Since mercury (II) ions are too small to elicit an immune response, they were coupled via glutathione (GSH) to the immunogenic carrier protein keyhole limpet hemocyanin (KLH). Several mice were immunised with this KLH-GSH-Hg immunoconjugate. Spleen cells of immunised mice were fused with myeloma cells. The resulting hybridoma cells were screened for the production of specific anti-Hg mabs. Five positive cells were detected. The hybridoma cell line K3C6 was adjusted to protein free medium; it produced mabs with high selectivity and sensitivity. A detection limit of 2.8 μg/L HgCl2 (= 2.1 μg/L Hg2+) was achieved with a non-competitive enzyme immunoassay (EIA). Cross-reactivities with other metals were below 1%. Measurement of spiked water samples with this EIA showed good correlation with results obtained by mass spectrometry with inductive coupled plasma (ICP-MS).

Three systems were tested for the optimization of a heterogenous non-competitive enzyme immunoassay (EIA) for the determination of Hg (II). The sensitivity of the non optimized Hg-EIA with a detection limit of 2.1 μg/L Hg (II) was improved by an avidin-biotin-complex (ABC) amplification system to a 2-fold lower detection limit (1.1 μg/L Hg (II)). A conventional competitive ElA with the competition reaction between bound and free Hg (II) for antibody (ab) binding sites yielded a detection limit of 1.0 μg/L Hg (II). Further improvement of sensitivity could be achieved by a competitive displacement EIA. In this case ab molecules bound to immobilized haptens are displaced in the next step by free Hg (II). The detection limit of the displacement approach is 0.4 μg/L Hg (II).

A novel method was developed for the fabrication of a surface plasmon resonance (SPR) sensor chip. A glass chip was covered with a gold layer which was then functionalized with gamma-aminopropylethoxysilane. Biomolecules could then be immobilized on the surface of the sensor using standard techniques. The sensor chip was found to be useful for highly sensitive SPR measurements using even smaller amounts of antibody than usual. The chip was found to be useful for the immobilization of anti-human serum albumin (anti-HSA) and anti-atrazine antibody, and was used for the SPR measurement of HSA and enzyme-labeled atrazine. The chip showed higher responses at both 10 and 100 mg l −1 HSA than a commercially available model. The relative standard deviation (RSD) of the responses was 7% for both concentrations of HSA ( n =8). A monoclonal antibody for atrazine was immobilized on the chip and 0.05 μg l −1 atrazine was measured with a RSD of 7% ( n =5). Atrazine was determined after addition to river water; 0.05 μg l −1 atrazine was detected with a very similar response as a standard atrazine solution of the same concentration.

The tendency of several pesticides to form non-extractable residues requires simple and fast screening methods. Immunochemical methods can meet this demand. They are based on selective and highly sensitive antibody binding to pesticide residues exposed on refractory organic substances (ROS). s-Triazines were used as an example to show the feasibility ofthis approach. It was proven that under laboratory conditions atrazine was bound to ROS. Antibody recognition pointed to the availability of free ethyl and isopropyl groups exposed at the non-extractable s-triazine residue. Therefore, it is suggested on the basis of antibody selectivities that binding of atrazine to ROS may take place by substitution of the chlorine residue. In the next step, aquatic ROS isolated from surface waters located in an agricultural area were examined for non-extractable s-triazines. ROS from a lake were found to contain bound residues, whereas none were detected in the creek samples. As an outlook the feasibility of serological characterization of ROS is discussed. The novel concept of serotyping is based on the fractionation of ROS by electrophoretic techniques followed by an immunoreaction with polyclonal antibodies directed against different ROS. When immunoelectrophoresis is used, precipitation lines can be compared and checked for identity and cross-reactivity. This approach can form the basis for classifying ROS and determine similarities with unknown ROS.

Reagent stability is an important issue in immunoassay technology. Enzyme immunoassays (EIA) for atrazine are used as an example for investigating the influence of different stabilizing agents on the activity of polyclonal and monoclonal antibodies (Ab), enzyme tracer (ET) as well as enzyme substrate after storage at different temperatures. When lyophilized Ab were stored for two weeks at 30°C, ca. 70% of the original activity in the EIA (lyophilized Ab stored at −20°C) was retained. In contrast, the activity of Ab-coated plates decreased very fast during storage at 30°C. This could be prevented by a post-coating step, especially with bovine serum albumin (BSA), polyvinyl alcohol (PVA) or a commercial solution (PAN/Coat™) as additives. The best stabilization of the ET was achieved with PVA (5%), BSA (1%), fetal calf serum (FCS, 0.5%) and two commercial stabilizing solutions. After storage at 4°C for 10 weeks, 100–175% of the activity of the control (ET stored at 4°C without additives) was observed. Activities similar to the control were also found after 10 weeks of storage at 30°C when two commercial stabilizing solutions were used. BSA (1%) was equally effective up to 6 weeks, subsequently the activity decreased to ca. 50% of the control. The enzyme substrate solutions (hydrogen peroxide and tetramethylbenzidine) should be stored separately. Under these conditions, they did not show any loss of activity after storage for two weeks at 30°C. The results obtained for the EIA were transferred to an immunofiltration assay, a membrane-based field test. Addition of BSA and PVA to the ET stored at 30°C led to the same color development as with the ET stored at 4°C without additives. The improved stability of the components of the EIA allows postal transfer without refrigeration and reliable performance of the tests in, and outside of the laboratory.

The wide variety of known and unknown xenoestrogenic compounds requires intelligent solutions to detect polluted environmental samples and identify the structure and concentration of the relevant xenoestrogens. In this article, a summary is given of several methods that are at present employed in the monitoring of xenoestrogens. Screening methods based on biological assays for the class-selective detection of xenoestrogens are reviewed. Furthermore, the analysis of known (xeno-)estrogens is summarized. Novel interfaces between biological assays and chromatography that facilitate the identification and quantification of xenoestrogens in complex samples are also discussed.

Gravitropic bending of fruiting bodies of Flammulina velutipes (Curtis) Karst. is based on the differential growth of the transition zone between stem and cap. Reorientation becomes visible as early as 2 h after displacing the fruiting body from the vertical to the horizontal position. It is preceded by a preferential accumulation of microvesicles within the hyphae on the lower side of the transition zone and related to an increase in the vacuolar compartment required for hyphal extension. A model made of a bundle of interconnected balloons is used to demonstrate that a differential volume increase at one flank is sufficient to bend the entire structure in the opposite direction. Gravitropic raising of intact stems or segments derived from the transition zone requires positional information which can be accomplished by three major, coordinated events: (i) gravisensing by the individual hyphae within the transition zone, (ii) unidirectional signalling by means of a soluble growth factor creating a vertical concentration gradient, and (iii) translation of the concentration signal into elongation growth.

The selectivity of immunoassay is limited by the cross-reactivity of antibodies to structurally related analytes. This becomes a drawback for applications that require discrimination of slightly different analytes. An approach to overcoming this problem is the application of antibody arrays that show differences in their affinity patterns. We have investigated this method using systematic modelling of multianalyte systems based on test-independent affinity parameters. A model system of anti-s-triazine antibodies and s-triazine derivatives has been investigated. The immunoassay is carried out in an indirect test format using an optical transducer for label-free monitoring of antibody binding at an immobilized hapten. The concentration of free antibody in equilibrium with the analyte is probed in a flow-through system. This format allows simple modelling of the response and assessment of the affinity constant from the calibration curve. The affinity patterns of five monoclonal antibodies and a recombinant single-chain fragment with respect to five s-triazine derivatives are determined by this method. An array of three antibodies is selected and the response pattern to mixtures of three analytes determined. Measured and calculated pattern correspond in principle, but systematic deviations are observed due to the perturbation of equilibrium during detection. The correlation of the true analyte concentration and the analyte concentrations predicted from the signal pattern using the affinity constants strongly depend on the selectivity and the affinity of the antibodies.

We present a method for the determination of affinity constants based on equilibrium binding between an analyte and an antibody in liquid phase by a heterogeneous phase detection scheme. Equilibrium concentration of free antibody binding sites was probed kinetically by direct optical detection of specific binding to an immobilised analyte derivative. The additional binding signal due to dissociation of the analyte-antibody complex during detection was minimised by the use of fast flow-through conditions. The concentration of free antibody binding sites was titrated by adding increasing analyte concentrations. The affinity constant was derived from the titration curve by a non-linear least square fit of a model function. The affinity of monoclonal triazine antibodies to several s-triazine pesticides and a relevant metabolite was investigated. Kinetic determination of equilibrium concentration of free binding sites was carried out by reflectometric interference spectroscopy (RIfS) using flow injection analysis. The capabilities of the model were investigated using different analyte-antibody pairs and various antibody concentrations. Both bivalent IgG and monovalent Fab fragments were used to compare different binding models. The applied model corresponds well to the titration curves for affinity constants of 10(7) M(-1) and higher. For lower affinity constants significant deviations due to dissociation of the analyte-antibody complex during detection were observed.