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Biomedical subjects

B Hock

Publications and source records attributed to B Hock.

At least 19 recordsLinked to original sources
PubMed2004

Surface plasmon resonance sensor for domoic acid based on grafted imprinted polymer.

A molecularly imprinted polymer (MIP) film for domoic acid (DA) was synthesised by direct photo-grafting onto a gold chip suitable for a surface plasmon resonance (SPR) based bioanalytical instrument system, the BIAcore 3000. The gold surface was first functionalised with a self-assembled monolayer of 2-mercaptoethylamine and subsequent carbodiimide chemistry was performed for covalent attachment of the photoinitiator, 4,4'-azobis(cyanovaleric acid). This ensured that the formation of the MIP thin film, comprising 2-(diethylamino) ethyl methacrylate as functional monomer and ethylene glycol dimethacrylate as cross-linker, occurred only at the surface level. Optimisation and control over the grafting procedure were achieved using contact angle measurements and atomic force microscope (AFM) imaging. The surface grafting resulted in the formation of thin and homogeneous MIP film with thickness of 40 nm. A competitive binding assay was performed with free DA and its conjugate with horseradish peroxidase, which was used as a refractive label. The sensor was evaluated for its sensitivity, cross-reactivity, and robustness by using a BIAcore 3000. Likewise, monoclonal antibodies acting as natural receptors for the toxin were studied with the same BIAcore system. Results of a comparison between the artificial and natural receptors are reported. In contrast to monoclonal antibodies, the regeneration of MIP chip did not affect its recognition properties and continuous measurement was possible over a period of at least 2 months.

Biosensing Techniques↗
PubMed2003

Recombinant antibodies for environmental analysis.

Initial steps of antibody engineering in the late eighties revolutionized the technology of antibody production, particularly in the area of immunotherapy and diagnostics. Hallmarks that seemed to be out of reach for a long time are now the state of the art, e.g. tailoring of antibodies to match particular needs or by-passing immunization by use of antibody libraries. Despite the apparent benefits of recombinant antibody technologies, this field has been opened up hesitantly for other applications. This review addresses the development of recombinant antibody synthesis in environmental analysis. Examples are given of the molecular evolution of pesticide antibodies and their application for the analysis of real samples.

Antibodies, Monoclonal↗
PubMed2002

Engineering receptors and antibodies for biosensors.

Biosensor sensitivity and selectivity depend essentially on the properties of the biorecognition elements to be used for analyte binding. Two principally different applications are considered, (1) effects monitoring with biological components as targets for bioeffective substances, among them endocrine disruptors; and (2) immunochemical analysis employing antibodies as binding proteins for a wide variety of analytes such as pesticides. Genetic engineering provides an elegant way not only for providing unlimited amounts of biorecognition molecules but also for the alteration of existing properties and the supplementation with additional functions. Instrumental applications were carried out with the optical sensor BIAcore. The first example deals with the characterization of receptors. For this purpose, the human estrogen receptor alpha was used. Binding studies were carried out with natural as well as xenoestrogens. An equilibrium dissociation constant K(d) of 2.3x10(-10) (M) was derived for 17beta-estradiol. A competition assay was performed with a bovine serum albumin (BSA)-17beta-estradiol conjugate, immobilized at the optical sensor surface, and the free estrogen. The signals obtained represent estradiol equivalents. This format was transferred to a microplate-based enzyme-linked receptor assay. It reached a detection limit of 0.02 microg l(-1) 17beta-estradiol and proved suitable for the detection of natural and synthetic estrogens as well as xenoestrogens in field studies. The second example is targeted at kinetic and affinity measurements of recombinant antibody fragments derived from antibody libraries with s-triazine selectivities. Different strategies for the synthesis of antibody fragment libraries, followed by the selection of specific antibody variants, were examined. An antibody library was derived from a set of B cells. Chain shuffling of the heavy and light chains provided the best binders. An enzyme linked immunosorbent assay (ELISA) was achieved for atrazine with an IC(50) of 0.9 microg l(-1) and a detection limit of 0.2 microg l(-1). The close relations between the optimization of recombinant antibodies by evolutionary strategies and genetic algorithms are considered.

Algorithms↗
PubMed2002

Vitellogenin levels in mussel hemolymph--a suitable biomarker for the exposure to estrogens?

Increased vitellogenin (vtg) levels in the blood of male fish are frequently used as an indicator of estrogenic exposure. Similar responses are expected for mussels, where the concentration of vtg-like proteins has been reported to depend on estrogens. To verify the role of hemolymph during vitellogenesis of mussels, the saltwater mussel Mytilus edulis and the freshwater mussel Anodonta cygnea were exposed to 17beta-estradiol (E2) and wastewater treatment plant effluents, known for their estrogenic potential. Gel electrophoresis did not reveal any significant induction (or repression) of plasma proteins compared to control plasma. Our results do not support the hypothesis that mussel hemolymph is a carrier of estrogen-dependent major egg-yolk precursors (vtg-like proteins). However, additional information on a 35+/-2-kDa hemolymph protein, previously reported to bind heavy metals, was obtained by high-resolution two-dimensional electrophoresis. It was resolved in a cluster of single proteins with properties that match the characteristics of a previously reported histidine-rich glycoprotein.

Animals↗
PubMed2002

Development of ELISA technique for the analysis of atrazine residues in water.

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.

Animals↗
PubMed2001

A new monoclonal antibody against vitellogenin from rainbow trout (Oncorhynchus mykiss).

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.

Animals↗
PubMed2001

Immunolocalization of non-extractable (bound) residues of pesticides and industrial contaminants in plants and soil.

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.

Antibodies↗
PubMed2000

Lectin ligands on human dendritic cells and identification of a peanut agglutinin positive subset in blood.

As only a few cell surface markers for dendritic cells (DC) have been identified to date, this study examined the expression of ligands for lectin on different human DC populations. The ability of Concanavalin A (Con A), Wheat Germ Agglutinin (WGA), peanut agglutinin (PNA), and Helix pomatia (HPA) to bind to cell lines and PBMC and DC populations was analyzed by flow cytometry and specificity of binding confirmed using inhibitory and noninhibitory sugars. The cell lines showed non-lineage-restricted binding with Con A and WGA, independent of sialidase treatment. HPA and PNA bound to a restricted number of lines, but showed broad reactivity after sialidase treatment. The peripheral blood mononuclear cells (PBMC) and directly isolated blood DC, activated CD83(+) blood DC, epidermal Langerhans cells (LC), and monocyte-derived DC (Mo-DC) showed strong binding of Con A and WGA, both before and after sialidase treatment. No HPA binding ligands were detected on PBMC populations, including directly isolated blood DC. Following sialidase treatment CD3(+), CD16(+), and a subset of CD19(+) lymphocytes bound HPA. The lectin PNA bound weakly to CD14(+) monocytes and a subpopulation of circulating DC that were HLA-DR(hi)CDw123 Dr(hi)CDw123(dim)/(neg)CD11c(+). The HLA-DR(mod)CDw123(hi)CD11c(neg) subpopulation did not bind PNA. Without sialidase treatment LC expressed both HPA and PNA ligands, but these were either absent on activated CD83(+) blood DC or weakly expressed on Mo-DC. Following sialidase treatment PBMC populations, activated CD83(+) blood DC, and Mo-DC became PNA positive. Thus human DC express several lectin ligands and PNA binding identifies a subset of blood DC. That may reflect discrete changes associated with stages of DC development or functional maturation.

Binding Sites↗
PubMed2000

Monoclonal antibody-based enzyme immunoassay for mercury(II) determination.

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%.

Absorption↗
PubMed1999

Chromosome mechanics of fungi under spaceflight conditions--tetrad analysis of two-factor crosses between spore color mutants of Sordaria macrospora.

Spore color mutants of the fungus Sordaria macrospora Auersw. were crossed under spaceflight conditions on the space shuttle to MIR mission S/MM 05 (STS-81). The arrangement of spores of different colors in the asci allowed conclusions on the influence of spaceflight conditions on sexual recombination in fungi. Experiments on a 1-g centrifuge in space and in parallel on the ground were used for controls. The samples were analyzed microscopically on their return to earth. Each fruiting body was assessed separately. Statistical analysis of the data showed a significant increase in gene recombination frequencies caused by the heavy ion particle stream in space radiation. The lack of gravity did not influence crossing-over frequencies. Hyphae of the flown samples were assessed for DNA strand breaks. No increase in damage was found compared with the ground samples. It was shown that S. macrospora is able to repair radiation-induced DNA strand breaks within hours.

Ascomycota↗
PubMed1998

PDZ-domain-mediated interaction of the Eph-related receptor tyrosine kinase EphB3 and the ras-binding protein AF6 depends on the kinase activity of the receptor.

Eph-related receptor tyrosine kinases (RTKs) have been implicated in intercellular communication during embryonic development. To elucidate their signal transduction pathways, we applied the yeast two-hybrid system. We could demonstrate that the carboxyl termini of the Eph-related RTKs EphA7, EphB2, EphB3, EphB5, and EphB6 interact with the PDZ domain of the ras-binding protein AF6. A mutational analysis revealed that six C-terminal residues of the receptors are involved in binding to the PDZ domain of AF6 in a sequence-specific fashion. Moreover, this PDZ domain also interacts with C-terminal sequences derived from other transmembrane receptors such as neurexins and the Notch ligand Jagged. In contrast to the association of EphB3 to the PDZ domain of AF6, the interaction with full-length AF6 clearly depends on the kinase activity of EphB3, suggesting a regulated mechanism for the PDZ-domain-mediated interaction. These data gave rise to the idea that the binding of AF6 to EphB3 occurs in a cooperative fashion because of synergistic effects involving different epitopes of both proteins. Moreover, in NIH 3T3 and NG108 cells endogenous AF6 is phosphorylated specifically by EphB3 and EphB2 in a ligand-dependent fashion. Our observations add the PDZ domain to the group of conserved protein modules such as Src-homology-2 (SH2) and phosphotyrosine-binding (PTB) domains that regulate signal transduction through their ability to mediate the interaction with RTKs.

3T3 Cells↗
PubMed1998

Tyrosine-614, the major autophosphorylation site of the receptor tyrosine kinase HEK2, functions as multi-docking site for SH2-domain mediated interactions.

HEK2 belongs to the family of EPH-related receptor tyrosine kinases (RTK) which are involved in axonal pathfinding and the formation of the embryonic body plan. The knowledge about intracellular pathways of signal transduction mediated by EPH-related receptors is still limited. Many of the known key players of cellular signalling contain Src homology 2 (SH2) domains, which recognize phosphotyrosine motifs in RTKs. Thus, we examined the interactions of various SH2-containing molecules like PLC-gamma1, rasGAP, p85 subunit of PI3-kinase, Src, Fyn, Crk, Nck, Grb2 and Shc with HEK2 using in vitro binding assays, immunoprecipitations and yeast Two-Hybrid assays. We found that rasGAP, Crk and Fyn bind in a SH2-dependent manner to autophosphorylated HEK2. rasGAP, which contains two SH2- and one SH3-domain, was shown to associate with its N-terminal SH2-domain to HEK2. Furthermore, we demonstrated that a single amino acid substitution (Y614F) clearly reduces the phosphotyrosine content of HEK2 and abrogates its ability to bind rasGAP, Crk and Fyn indicating that this residue functions as major phosphorylation and multi-docking site. The conservation of this predicted binding site among various EPH-related RTKs provides evidence that Fyn, Crk and rasGAP are key players in signal transduction of at least a subset of these receptors.

Binding Sites↗
PubMed1998

Nef proteins of distinct HIV-1 or -2 isolates differ in their binding properties for HCK: isolation of a novel Nef binding factor with characteristics of an adaptor protein.

The Nef gene of the human and simian immunodeficiency viruses HIV and SIV has been implicated in pathogenicity; however, the mechanism by which Nef induces disease is still unknown. An impact on signal transduction in cells has been suggested by the interaction of Nef from an HIV-1 strain and tyrosine kinases like HCK and LCK as well as serine/threonine kinases. We have confirmed the binding of HCK to HIV-1 subtype B Nef and demonstrated an equally strong interaction with a subtype E Nef protein but weaker binding to Nef of HIV-2 subtype A (HIV-2D194). No binding, however, was observed to HIV-2 subtype B Nef (HIV-2D205). Instead, this protein bound to a novel cellular protein, Nefin 1, with characteristics of an adaptor protein and strong expression in all human hematopoietic tissues. Nefin 1 binds through an amino-terminal domain, which is related to SH3 domains. For interaction of Nef with Nefin 1, the PxxP motif and the three-dimensional conformation of the molecule appear necessary. In conclusion, this study demonstrates that Nef proteins of divergent strains of HIV-1 and HIV-2 may use different elements of signal transduction pathways for the induction of pathogenicity in vivo.

Adaptor Proteins, Signal Transducing↗
PubMed1998

Immunoenzymatic determination of atrazine in rat tissue samples.

In the present study our objective was to adapt a competitive enzyme immunoassay (EIA) for the determination of atrazine in animal tissues (liver and kidney). Female Wistar rats were administered through gavage with a single dose of atrazine (500 or 1000 mg/kg body weight) and sacrificed 24, 48 h and 7 days following the exposure. Using liver tissue samples the extraction procedure was improved. The atrazine concentrations measured in the liver were higher than those found in the kidney, but both can be ranked as low compared with the amount of the administered doses. These data confirm that tissue retention is minimal. Further studies are necessary in order to make an overall evaluation concerning the amount of atrazine retained in the organism. We consider that the competitive EIA may be a promising technique for epidemiological screening of pesticides.

Administration, Oral↗
PubMed1998

The waveguide Mach-Zender interferometer as atrazine sensor.

Immunoanalytical techniques used in combination with a highly sensitive Mach-Zender waveguide sensor give a device that is capable of fast on-line monitoring of immunoreactions. The on-line monitoring is especially attractive when the sensor can be used as an environmental probe. It is demonstrated that low concentrations of atrazine can be measured with a sensitivity around the EC limit of 100 ng/L. These measurements are performed with an inhibition technique. In combination with "a dynamic slope method" one complete measurement is done within 10 min. Sensor measurements show the same sensitivity and errors as parallel ELISAs.

Atrazine↗
PubMed1998

Continuous flow immunosensor for atrazine detection.

The hapten atrazine was detected under continuous flow conditions using a micro-column which contained immobilized monoclonal antibodies (Ab) against atrazine and atrazine labeled with alkaline phosphatase (An*). The equilibrium of the antibody-hapten system, was achieved by a continuous flow of the tracer An* through the micro-column containing the immobilized antibodies. The activity of the tracer was monitored continuously, after the micro-column, by an amperometric detector using p-hydroquinone phosphate as substrate. When pulses of unlabeled atrazine (An) were added to the An* flowing continuously through the micro-column, An* bound to the antibody was displaced, with a consequent change of the detector signal. By this method atrazine concentrations in the range 9-180 micrograms/l were monitored under conditions of continuous operation. Since the equilibrium condition for the system Ab-An* was continuously restored by the flow of An* through the micro-column the regeneration of the antibody was not required.

Animals↗
PubMed1998

In vitro analysis of xenoestrogens by enzyme linked receptor assays (ELRA).

Receptor binding assays are a modern approach to the effects-related analysis of xenoendocrines. Human estrogen receptors are used to develop an analytical tool for the detection of estrogenic substances in environmental samples. A sensitive Enzyme Linked Receptor Assay (ELRA) was developed in a microwell plate format. The receptor assay is based on similar principles as competitive immunoassays (ELISA). However, receptor binding always implies a biological effect, either agonistic or antagonistic. The choice of suitable tracers or hapten conjugates is an important step in assay development. For this purpose and for the determination of receptor-affinities of relevant xenoestrogens, a surface plasmon resonance (SPR) biosensor (BIAcore) was used for binding studies with immobilized receptors. Results with commercially available hapten-conjugates (estradiol-BSA) show a direct correlation between the amount of immobilized estradiol receptor and the amount of bound hapten-conjugate. Based on these BIAcore experiments an ELRA was developed. The calibration curves show a detection limit of 0.1 microgram/l for 17 beta-estradiol. Cross-reactivities of different steroids and xenoestrogens are reported.

Biosensing Techniques↗