Quantum dot loaded liposomes as fluorescent labels for immunoassay.

Liposomes loaded with water-soluble and water-insoluble quantum dots (QD) were for the first time applied as labels in different heterogeneous immunoassays for the determination of food contaminants, using mycotoxin zearalenone (ZEN) as a model. A great deal of work was devoted to the optimal choice of phospholipids for the liposomes preparation and to the factors which are important for the stability and size of obtained liposomes. Thin-film hydration and reverse-phase evaporation techniques were evaluated in terms of stability of the obtained liposomes and their efficiency for QD loading. Conjugation of liposomes with proteins and the influence of cross-linkers to the nonspecific interaction of the obtained liposomes with the surface of microtiter plates and cartridges were investigated and 3-(2-pyridyldithio)propionic acid N-hydroxysuccinimide ester was found as the optimal cross-linker. The limits of detection (LOD) for ZEN of fluorescence-labeled immunosorbent assays were 0.6 µg kg(-1), 0.08 µg kg(-1), and 0.02 µg kg(-1), using QD, liposomes loaded with water-soluble QD, and water-insoluble QD, respectively. Similarly, the developed qualitative on-site tests using the different QD labels and taking into account the EU maximum residues level for ZEN in unprocessed cereals showed cutoff levels of 100, 50, and 20 µg kg(-1).

Automated regenerable microarray-based immunoassay for rapid parallel quantification of mycotoxins in cereals.

An automated flow-through multi-mycotoxin immunoassay using the stand-alone Munich Chip Reader 3 platform and reusable biochips was developed and evaluated. This technology combines a unique microarray, prepared by covalent immobilization of target analytes or derivatives on diamino-poly(ethylene glycol) functionalized glass slides, with a dedicated chemiluminescence readout by a CCD camera. In a first stage, we aimed for the parallel detection of aflatoxins, ochratoxin A, deoxynivalenol, and fumonisins in cereal samples in a competitive indirect immunoassay format. The method combines sample extraction with methanol/water (80:20, v/v), extract filtration and dilution, and immunodetection using horseradish peroxidase-labeled anti-mouse IgG antibodies. The total analysis time, including extraction, extract dilution, measurement, and surface regeneration, was 19 min. The prepared microarray chip was reusable for at least 50 times. Oat extract revealed itself as a representative sample matrix for preparation of mycotoxin standards and determination of different types of cereals such as oat, wheat, rye, and maize polenta at relevant concentrations according to the European Commission regulation. The recovery rates of fortified samples in different matrices, with 55-80 and 58-79%, were lower for the better water-soluble fumonisin B1 and deoxynivalenol and with 127-132 and 82-120% higher for the more unpolar aflatoxins and ochratoxin A, respectively. Finally, the results of wheat samples which were naturally contaminated with deoxynivalenol were critically compared in an interlaboratory comparison with data obtained from microtiter plate ELISA, aokinmycontrol® method, and liquid chromatography-mass spectrometry and found to be in good agreement.

Low molecular weight, non-peptide fibrinogen receptor antagonists.

The tetrapeptide H-Arg-Gly-Asp-Ser-OH (1) (RGDS), representing a recognition sequence of fibrinogen for its platelet receptor GP IIb-IIIa (integrin alpha IIb beta 3), served as lead compound for the development of highly potent and selective fibrinogen receptor antagonists. Replacement of the N-terminal arginine by p-amidinophenylalanine or the Gly moiety by m-aminobenzoic acid led to compounds which are superior to the lead peptide with regard to activity and selectivity for GP IIb-IIIa vs the closely related vitronectin receptor alpha v beta 3. By random screening [(p-amidinobenzenesulfonamido)ethyl]-p-phenoxyacetic acid derivatives have been identified as fibrinogen receptor antagonists. Further structure-activity relationship studies culminated in the preparation of N-[N-[N-(p-amidinobenzoyl)-beta-alanyl]-L-alpha-aspartyl]-3-phenyl-L- alanine (29h, Ro 43-5054) and [[1-[N-(p-amidinobenzoyl)-L-tyrosyl]-4-piperidinyl]oxy]acetic acid (37f, Ro 44-9883), which exhibit very high activity as platelet aggregation inhibitors (IC50s 0.06 and 0.03 microM, respectively, human PRP/ADP) as well as marked selectivity for GP IIb-IIIa vs alpha v beta 3. Since the activity of 37f in dogs declines according to a two-compartment model with an initial phase having a t1/2 of 8 min and a second phase with a t1/2 of 110 min, this compound is a suitable candidate for the development as iv platelet inhibitor.

Orally active fibrinogen receptor antagonists. 2. Amidoximes as prodrugs of amidines.

The potent and selective GP IIb-IIIa antagonist lamifiban (1, Ro 44-9883) is currently in clinical development as an injectable antithrombotic agent for treating and preventing acute coronary syndromes. However, for secondary prevention of thrombotic occlusions, orally active inhibitors are needed. By means of a prodrug strategy, the modest oral absorption of 1 in mice was improved by a factor of 9. In addition, these studies demonstrated that an amidoxime group can serve as a prodrug functionality for an amidino group. Application of this principle to the structurally related amidino carboxylate 13 led to the amidoxime ester 18 which was absorbed approximately 20 times better, after oral administration to mice, than 13. Due to the modification of the amidino group as well as of the carboxylate group, 18 completely lost its ability to interact with purified platelet GP IIb-IIIa. After oral administration of 18 to rats, dogs, and rhesus monkeys, the bioavailability of the active derivative 13 was 26 +/- 5, 25 +/- 6, and 33 +/- 6%, respectively, and the elimination half-life was 4.1 +/- 1.7, 11.4 +/- 1.1, and 5.1 +/- 1.4 h, respectively. On the basis of these properties, the orally active 18 (Ro 48-3657), a double prodrug of the potent and selective non-peptide GP IIb-IIIa antagonist 13 (Ro 44-3888), was selected as clinical candidate for evaluation as a prophylactic agent in patients at high risk for arterial thrombosis.

Determination of 1-nitropyrene with enzyme-linked immunosorbent assay versus high-performance column switching technique.

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.

On-line coupling of sol-gel-generated immunoaffinity columns with high-performance liquid chromatography.

The paper demonstrates the possibility to use sol-gel-generated immunoaffinity columns as selective sample preparation step in on-line combination with HPLC. In the past sol-gel-generated immunoaffinity columns have only been included in off-line sample preparation schemes. Compared with conventional RP-materials on-line coupling of sol-gel-generated silica matrices with a pore structure designed to retain antibodies poses additional problems caused by their lower pressure tolerance and by the necessity to match the mobile phases not only to take into account the chromatographic properties but also the conformational stability of the antibodies. These problems have been overcome by an on-line system which can be regarded as a prototype for similar systems which exploit the selectivity of sol-gel immunoaffinity columns. The system consists of a sol-gel-generated immunoaffinity column coupled to an RP enrichment column and an analytical column. The practicality of such systems is demonstrated using the example of anti-pyrene immunoaffinity columns applied for the determination of pyrene in aqueous solutions.

Determination of 1-nitropyrene in herbs after selective enrichment by a sol-gel-generated immunoaffinity column.

Using the determination of 1-nitropyrene as an example the paper demonstrates the advantages of including a highly selective sol-gel-generated immunoaffinity column in the sequence of clean-up steps necessary to determine haptens in complex matrices. The sol-gel method to immobilise antibodies enlarges the variety of immunoaffinity columns available and leads to mechanically stable columns with constant retention characteristics. The sample preparation scheme proposed combines acetonitrile extraction, size-exclusion and immunoaffinity chromatography. 1-Nitropyrene is then separated by reversed-phase HPLC from interfering compounds and determined after catalytic on-line reduction to the corresponding amine by spectrofluorimetry. Concentrations in the range from 0.1 to 1.4 microg/kg 1-nitropyrene were detected in herbs.

Optimization of reverse hybridization in microplates coated with rRNA targeted oligonucleotide probes.

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 nonchemically 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 nested array of rRNA targeted probes for the detection and identification of enterococci by reverse hybridization.

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.

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Magnetic nanogold microspheres-based lateral-flow immunodipstick for rapid detection of aflatoxin B2 in food.

A novel membrane-based lateral-flow immunodipstick assay was developed for the fast screening of aflatoxin B(2) (AFB(2)) as a model compound in food samples. The detector reagent consisted of magnetic nanogold microspheres (MnGMs) with nano-Fe(2)O(3) particles as core and gold nanoparticles as shell, and bio-functionalized with monoclonal anti-AFB(2) antibodies. Manually spotted AFB(2)-bovine serum albumin conjugates (AFB(2)-BSA) and goat anti-mouse IgG on nitrocellulose membrane were used as test and control lines, respectively. As the major advantage, experimental results indicated that the visual detection limit (cutoff value) of the MnGM-based dipstick immunoassay with 0.9 ng/ml AFB(2) was about threefold lower compared to a conventional immunodipstick test using gold nanoparticles as detection reagent. Qualitative results (yes/no) could be obtained within 15 min without expensive equipment. The assay was evaluated with AFB(2) spiked or naturally contaminated samples (n=8), including peanuts, hazelnuts, pistacia and almonds, receiving excellent correspondance with results from high performance liquid chromatography (HPLC). Most importantly, the assay gave no false negative results. By controlling the target antibody this assay can be easily extended for use with other food relevant toxins and thus represents a versatile detection method.

Selective trace analysis of diclofenac in surface and wastewater samples using solid-phase extraction with a new molecularly imprinted polymer.

A new molecularly imprinted polymer (MIP) for trace analysis of diclofenac in environmental water samples was prepared by a non-covalent protocol in which diclofenac was used as a template molecule. Diclofenac is a member of the class of drugs termed non-steroidal anti-inflammatory drugs (NSAIDs) which belong to the most frequently detected pharmaceuticals in the water-cycle in Europe. The MIP was synthesized using 2-vinylpyridine (2-VP) and ethylene glycol dimethacrylate (EGDMA) as a functional monomer and cross-linker, respectively, and bulk thermal polymerization method. (1)H NMR spectroscopy was used to study the interaction between diclofenac and 2-VP mixed in toluene-d(8) in pre-polymerization complex. Two non-covalent bonds were formed i.e. ionic interaction and hydrogen bonding. The binding characteristics of the MIP and diclofenac were evaluated using equilibrium binding experiments. Scatchard plot analysis revealed that two classes of binding sites were formed with dissociation constants of 55.6 micromol L(-1) and 1.43 mmol L(-1), respectively. Various parameters affecting the extraction efficiency of the polymers have been evaluated to achieve the selective preconcentration of diclofenac from aqueous samples and to reduce non-specific interactions. This resulted in an MISPE-LC/DAD method allowing the direct extraction of the analyte from sample matrix with a selective wash using dichloromethane/acetonitrile (94:6, v/v) followed by elution with dichloromethane/methanol (85:15, v/v). The recovery of a 100 ng diclofenac standard spiked into 200 mL of blank surface water was 96%, with good precision (RSD=3.3%, n=3). The MISPE was demonstrated to be applicable to the analysis of diclofenac in raw influent and final effluent wastewater samples from sewage treatment plant and revealed diclofenac concentrations of 1.31+/-0.055 microg L(-1) (n=3) and 1.60+/-0.049 microg L(-1) (n=3), respectively. Yielded results were in good agreement with the corresponding LC/TIS/MS/MS data obtained by an independent laboratory which were 1.40 and 1.50 microg L(-1) for influent and effluent samples.

Assessment of exposure to 2,4-dichlorophenoxyacetic acid in the chemical industry: results of a five year biological monitoring study.

Data on individual exposure to 2,4-dichlorophenoxyacetic acid (2,4-D) in herbicide production plants are limited. Hence, the urinary excretion of this herbicide was measured during a five year (1985-1989) biological monitoring study of 27 men and 18 women employees exposed during the production and formulation of 2,4-D and related sodium and dimethylamine salts. In separate studies, specimens of urine were collected in the morning, or during the last three hours of a working shift, or over a 24 hour period (1200 to 1200 or 0800 to 0800) and were analysed by an immunochemical method (2,4-D radioimmunoassay (RIA)). Urinary 2,4-D concentrations varied within a large scale from only a few micrograms/l to several 10s of mg/l. During a week, herbicide excretion increased, culminating on Friday. At the weekend, when no work was done, 2,4-D elimination decreased but did not return to zero in any case. After an interruption of exposure for about three weeks, urinary 2,4-D was no longer detectable. About five days after restarting work, body concentrations had built up again. Measurements of 2,4-D concentrations in air at different work-places showed that herbicide concentrations did not exceed 0.5 mg/m3. As well as inhalation, dermal 2,4-D absorption seemed to play an important part in total uptake of herbicide. Furthermore, a strong correlation was found (r = 0.9628) between 2,4-D urinary concentration, adjusted for endogenous creatinine, and the estimated amount of absorbed herbicide. Estimated absorbed doses were, in most cases, well below 0.1 mg 2,4-D/kg body wt/day. Sometimes this concentration was greatly exceeded. Thyroid hormone concentrations in blood were measured as well. No notable abnormalities were found. Exposed subjects were also typed for histocompatibility locus antigens (ABC antigens). The immunochemical determination of 2,4-D in specimens of urine proved to be a simple, cost effective, and non-invasive method to measure human exposure.

[Family planning in the guidelines of the National Congress]. / Planejamento familiar em pauta no Congresso Nacional.

PIP: In Brazilian Congress, the importance of family planning has, for historical reasons, been confused with controlling population growth. The question is mostly related to the iniquitous distribution of wealth rather than overpopulation. The populations of developed countries consume energy, primary materials, and foodstuffs for a comfortable living, however, a large part of the populations in countries on the peripheries barely survive with incomes below the poverty line. In Brazil, the richest 10% possess half of the national wealth, and the remaining 90% are left with the other half. Most of all, the lack of access to produced wealth is the reason why the majority do not have access to health care, education, leisure, information, culture, and the conditions of living with dignity. The adoption of contraceptive methods is intimately associated with the level of culture and information of the population. The majority of women desire to use some kind of contraceptive method. It is imperative that the state grant to the women and men who so desire access to the materials and necessary information in order to choose the number of children they desire. The Ministry of Health has run for almost a decade a program for maternal health (PAISM), although it has not been fully implemented, and the benefits of this program have remained inaccessible to large numbers of women, especially in the large metropolises. In Brazil, about 30% of women of reproductive age undergo sterilization, as opposed to only 7% in developed countries. Those who cannot afford it give birth to children who grow up without education or medical care and become immersed in violence to fight for their survival.^ieng

Biological monitoring of 2,4-dichlorophenoxyacetic acid-exposed workers in agriculture and forestry.

This is a report on the application of a radioimmunoassay (RIA) for the herbicide 2,4-dichlorophenoxyacetic acid (2,4-D) in biological monitoring of occupationally exposed sprayers. Urinalysis was conducted on two workers involved in spraying 2,4-D sodium salt solution with car mounted ground rigs in agriculture and on the pilot and the mixer-loader of a helicopter crew applying 2,4-D dimethylamine salt for brush control in forestry. All sprayers showed detectable quantities of 2,4-D in morning urine samples voided over 4 or 6 days in the post-spraying period. The highest 2,4-D urinary concentrations of about 2.5 ppm could be measured in a car driver on the 3 day after exposure. The 2,4-D level in urine was much lower in forestry workers, with 0.365 ppm for the mixer-loader and 0.052 ppm for the pilot on the 1 day after spraying. Urinary 2,4-D concentrations in the agricultural study were adjusted for endogenous creatinine and, when normalized for body weight, resulted in a total 2,4-D uptake of about 5.7 or 84.9 micrograms/kg body wt. for a single spraying operation. Thus, the calculated absorbed dose was less than the NOAEL (no observed adverse effect level) of 10 mg/kg body weight per day by a large margin of safety. Further, the calculated amounts excreted seemed to be sufficiently reliable to be used for assessing the risk of human exposure to 2,4-D. However, more studies are desirable for confirmation of the association of urinary 2,4-D and creatinine excretion. Clearance occurs with a 12-h to 22-h half-life.

Oral and dermal application of 2,4-dichlorophenoxyacetic acid sodium and dimethylamine salts to male rats: investigations on absorption and excretion as well as induction of hepatic mixed-function oxidase activities.

Absorption and urinary excretion of 2,4-dichlorophenoxyacetic acid sodium (sodium 2,4-D) and 2,4-dichlorophenoxyacetic acid dimethylammonium (2,4-DMA) salts were examined after single oral and mid-dorsal skin applications of the herbicides to male rats. Doses of 2.6 mg 2,4-D/kg body wt (sodium 2,4-D) and 1.9 mg 2,4-D/kg body wt (2,4-DMA) were applied. Quantitatively, with both salts, most of the orally administered herbicide (over 90%) was excreted in urine within 28 h. However, 2,4-D urinary peak concentrations were measured 4.5 and 20.5 h after dosing with 2,4-DMA and sodium 2,4-D, respectively. Additionally, the volume of urine in the oral study was significantly increased with both salts when compared with the controls or the dermal exposure. After topical application, 2,4-D absorption was much lower than in the oral study. Urinary excretion only reached about 10 and 15% of the applied dose for sodium 2,4-D and 2,4-DMA, respectively, by 5 days post-treatment. Further, we found some elevations in hepatic cytochrome P-450 activities. Ethylmorphine N-demethylase was only slightly induced by the herbicides while ethoxyresorufin O-deethylase activity was increased nearly 2-fold by sodium 2,4-D.

[Organic trace element pollution in water--a potential health risk for long-term dialysis patients]. / Organische Spurenverunreinigungen im Wasser--ein potentielles Gesundheitsrisiko für Dauerdialysepatienten.

During clinical hemodialysis patients blood is exposed in general 3 times every week to about 150 l of dialysate. The quality of the water used to prepare dialysate solution is therefore of great importance. A potential health hazard exists especially for those contaminants which are protein-bound in the blood. As a consequence, even if present in dialysate in lower concentrations than in blood that substances may still cross the dialysis membrane and accumulate in the body to toxic levels. The present paper describes the behaviour of the herbicide 2,4-dichlorophenoxyacetic acid that is known for its high plasma protein binding, during in vitro study using standard dialysis devices. The quantification of the herbicide in dialysate and plasma was performed with a radioimmunoassay.

[Immunoassays. Performance efficient methods of analysis for environmental toxicologic research and practice]. / Immunoassays. Leistungsfähige Analysenmethoden für die umwelttoxikologische Forschung und Praxis.

A review is presented which summarizes the development and application of immunoassays for pesticides and other environmental pollutants. Especially, in the last ten years an increased activity in that field can be noted. With the production of monoclonal antibodies an essential prerequisite is realized for standardization and commercialization of that methods. Potential application fields are routine measurements of environmental probes, biomonitoring in occupational hygiene and in epidemiological studies, determination of compounds difficult to analyze by classical analytical methods, application in chemical damages, control of hazardous waste deposits as well as for quality control of biotechnological products. Related research should be forced also in the GDR.