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At least 1,351 records · Page 75Linked to original sources
PubMed1998

A high-throughput, microtiter plate assay for paralytic shellfish poisons using the saxitoxin-specific receptor, saxiphilin.

An isoform of the paralytic shellfish poison (PSP)-specific receptor saxiphilin, from the tropical centipede Ethmostigmus rubripes, was used as the basis for a radiometric, high-throughput, microtiter plate assay for this group of toxins. Characterization of the assay revealed that it was able to detect several representatives from the various structural PSP subgroups and yet was insensitive toward tetrodotoxin. To test the utility of the assay as a seafood toxin-monitoring tool, the assay was subjected to a variety of marine organism extracts, some of which were known to contain PSPs, and whole extract toxicity expressed as STX equivalents (STXeq) was measured by two methods: First, by comparison of values from a screening assay with a standard STX inhibition curve and, second, for highly active extracts, by calculation using the IC50 from a full inhibition curve of the extract. For extracts which could be quantified by both methods, there was almost 100% correlation between the derived values. STXeq derived by both methods from the bioassay highly correlated with absolute toxin quantities from HPLC analysis.

Amphibian Proteins↗
PubMed2004

Fluorescence polarization assays for high-throughput screening of neuropeptide FF receptors.

We have developed the first fluorescence polarization assays of human neuropeptide FF2 receptors in 384-well microtiter plates. Assays are completed in a single well with no transfer, separation, or wash steps. The performance is suitable for high-throughput drug screening applications with regard to speed of analysis, magnitude of displaceable signal, precision, and sensitivity of various reagents. The rank order of potency of agonists and antagonists agrees well relative to the published radiometric filtration assays: DMe NPFF > NPFF > frog PP (Rana temporaria pancreatic polypeptide) > PQRFamide > BIBP 3226. The effect of highly colored compounds is very small on the polarization signal up to micromolar concentrations. The method serves as a simple and fast alternative to radioligand binding assays of antiobesity drug candidates related to NPFF receptors.

Animals↗
PubMed2005

Automatic classification and pattern discovery in high-throughput protein crystallization trials.

Conceptually, protein crystallization can be divided into two phases search and optimization. Robotic protein crystallization screening can speed up the search phase, and has a potential to increase process quality. Automated image classification helps to increase throughput and consistently generate objective results. Although the classification accuracy can always be improved, our image analysis system can classify images from 1,536-well plates with high classification accuracy (85%) and ROC score (0.87), as evaluated on 127 human-classified protein screens containing 5,600 crystal images and 189,472 non-crystal images. Data mining can integrate results from high-throughput screens with information about crystallizing conditions, intrinsic protein properties, and results from crystallization optimization. We apply association mining, a data mining approach that identifies frequently occurring patterns among variables and their values. This approach segregates proteins into groups based on how they react in a broad range of conditions, and clusters cocktails to reflect their potential to achieve crystallization. These results may lead to crystallization screen optimization, and reveal associations between protein properties and crystallization conditions. We also postulate that past experience may lead us to the identification of initial conditions favorable to crystallization for novel proteins.

Algorithms↗
PubMed2001

High-throughput biopolymer desalting by solid-phase extraction prior to mass spectrometric analysis.

In the last 10 years mass spectrometry (MS) has become an important method for analysis of peptides, proteins and DNA. It was recently utilized for accurate high-throughput protein identification, sequencing and DNA genotyping. The presence of non-volatile buffers compromises sensitivity and accuracy of MS biopolymer analysis; it is essential to remove sample contaminants prior to analysis. We have developed a fast and efficient method for desalting of DNA oligonucleotides and peptides using 96-well solid-phase extraction plates packed with 5 mg of Waters Oasis HLB sorbent (Waters, Milford, MA, USA). This reversed-phase sorbent retains the biopolymer analytes, while non-retained inorganic ions are washed out with pure deionized water. DNA oligonucleotides or peptides are eluted using a small amount (20-100 microl) of acetonitrile-water (70:30, v/v) solution. The SPE desalting performance meets the requirements for MS applications such as protein digest analysis and DNA genotyping.

Biopolymers↗
PubMed1998

Optimization and automation of fluorescence-based DNA hybridization for high-throughput clone mapping.

Large-scale hybridization-based genome mapping projects, such as the production of sequence-ready physical clone maps, call for robust and cheap DNA labeling techniques that are amenable to automation. We routinely use a high-throughput protocol based on fluorescence detection. DNA probes are labeled via polymerase chain reaction (PCR) amplification with primers that are digoxigenin-modified at their 5' ends. Alternatively, digoxigenin-labeled dUTP is incorporated in a random hexamer priming reaction. Hybridization takes place in small volumes by sandwiching the probe between filters and plastic sheets. A fluorescence signal is produced by the activity of alkaline phosphatase attached to an anti-digoxigenin antibody upon the addition of AttoPhos substrate. Signals are directly detected with a charge-coupled device (CCD) camera and scored by an image data analysis system. DNA filters can be reused at least 40 times without loss of data quality. Significant advantages compared to radioactive techniques are the reduced health risk, enabling highly parallel processing; the production of spot signals uniform in size and intensity, which is essential for efficient image analysis; and a cost reduction of about 70%.

Alkaline Phosphatase↗
PubMed2006

Development of a high-throughput membrane-array method for molecular diagnosis of circulating tumor cells in patients with gastric cancers.

Recently several noninvasive methods have been employed to detect circulating tumor cells (CTCs) in cancer patients. In this study, we have developed a highly sensitive, high-throughput colorimetric membrane-array method that was designed to detect a panel of mRNA markers including human telomerase reverse transcriptase (hTERT), cytrokeratin-19 (CK-19), carcinoembryonic antigen (CEA) and mucin 1 (MUC1) mRNA for the presence of CTCs in the peripheral blood of patients with gastric cancer (GC). Digoxigenin-labeled cDNA targets synthesized following total RNA isolation from peripheral blood samples of 64 GC patients and 80 healthy individuals were subjected to membrane-array hybridization. The results showed that membrane array could positively detect 5 cancer cells/ml of peripheral blood in GC cell-dilution experiments. The sensitivity, specificity and diagnostic accuracy for hTERT, CK-19, CEA and MUC1 mRNA ranged from 78.1% to 82.8%, 76.3% to 85% and 81.3% to 83.3%, respectively. Both CEA and MUC1 mRNA expression was correlated significantly with all malignant biological properties of GC, such as macroscopic type, depth of tumor invasion, lymph-node metastasis, TNM stage and coexisting distant metastasis (all p < 0.05). Using these 4 markers in combination, the sensitivity, specificity and diagnostic accuracy of membrane array were raised to 89.1%, 91.3% and 90.3%, respectively. The expression of all 4 mRNA markers was an independent predictor for postoperative recurrence/metastasis. GC patients with the expression of all the 4 mRNA markers showed a poorer survival rate than those without the expression of any 1 mRNA marker (p = 0.0223). These findings demonstrated that our membrane-array method could detect CTCs in the circulation of GC patients with considerably high sensitivity and specificity. The identification of CTCs in the peripheral blood may be useful in the auxiliary cancer diagnostics or postoperative surveillance of GC patients for recurrence/metastasis.

Adult↗
PubMed2002

High-throughput approaches for the discovery and optimization of new olefin polymerization catalysts.

The discovery of new olefin polymerization catalysts is currently a time-intensive trial-and-error process with no guarantee of success. A fully integrated high-throughput screening workflow for the discovery of new catalysts for polyolefin production has been implemented at Symyx Technologies. The workflow includes the design of the metal-ligand libraries using custom-made computer software, automated delivery of metal precursors and ligands into the reactors using a liquid-handling robot, and a rapid primary screen that serves to assess the potential of each metalligand-activator combination as an olefin polymerization catalyst. "Hits" from the primary screen are subjected to secondary screens using a 48-cell parallel polymerization reactor. Individual polymerization reactions are monitored in real time under conditions that provide meaningful information about the performance capabilities of each catalyst. Rapid polymer characterization techniques support the primary and secondary screens. We have discovered many new and interesting catalyst classes using this technology.

Journal Article↗
PubMed2005

High throughput screening of meningioma biomarkers using a tissue microarray.

Meningiomas are histologically and clinically diverse CNS neoplasms with few available immunohistochemical markers of differentiation and progression. Therefore, we investigated a panel of potentially useful meningioma-associated biomarkers using high throughput tissue microarray immunohistochemistry (TMA-IHC) with a TMA that includes 9 hemangiopericytomas (HPCs) and 41 meningiomas spanning all grades, as well as two subsets of atypical meningiomas, stratified according to clinical behavior. Antibodies utilized were progesterone receptor (PR), epithelial membrane antigen (EMA), cathepsin D, E-cadherin, platelet derived growth factor (PDGF) receptor beta, PDGF BB ligand, survivin, epithelial growth factor receptor (EGFR), and vascular endothelial growth factor (VEGF). In most cases, frequencies of tumor positivity were similar to those previously reported using whole section IHC. EMA, E-cadherin, and PDGFR-beta staining patterns distinguished the anaplastic meningiomas from the HPCs (P < 0.001, P = 0.02, P = 0.015, respectively). As in prior studies, PR and cathepsin D expression were inversely proportional to tumor grade. However, PR and EGFR were also differentially expressed between symptomatic, surgically resected benign meningiomas and incidental meningiomas found at autopsy. We conclude that (1) TMA-IHC is an accurate and efficient way to rapidly assess biomarkers in meningeal tumors, (2) EMA, E-cadherin, and PDGFR-beta are useful in distinguishing anaplastic meningiomas from HPCs, and (3) the expression patterns for incidental meningiomas differ slightly from their surgically resected symptomatic counterparts.

Biomarkers, Tumor↗
PubMed1998

Synthesis of sialyl Lewis X pentasaccharide analogue for high-throughput screening of selectin blockers.

We have developed an effective synthesis of sLe(x) pentasaccharide glycolipid analogue 2. As a part of application of sLe(x) pentasaccharide glycolipid 2 synthesized here, we have investigated the construction of a high-through-put screening system for discovery of selectin blockers. As a result, it was found that compound 2 was a useful ligand for in vitro ELISA assay and could be an important material for high-throughput screening of selectin blockers.

Carbohydrate Conformation↗
PubMedDate not supplied

High-throughput selection for heterogeneous catalysts.

A catalyst library of 80 samples with different mass ratios of rare earth elements, cobalt (Co), cerium (Ce), and indium (In), was prepared by impregnation of a fresh HZSM-5 zeolite support. A high-throughput detection setup, based on UV absorption spectroscopy, was developed for heterogeneous catalyst selection. The catalytic properties of the library were tested in the selective catalytic reduction of NO by methane at 673 K. Among the Co/Ce series, the catalyst with Co/Ce = 2:1 and Co/H-ZSM5 = 2.5% has shown remarkable efficiency (up to 78%). In the Ce/In series, the reactivity of the catalyst with the support composition of Ce/In = 1:1 and Ce/H-ZSM5 = 2.0% was < or = 88%. Our initial experiments definitely indicated that this simple and inexpensive multichannel setup can be applied for the selection of other heterogeneous catalysts. According to the variation of the UV light intensity, resulting from the absorption of the reactant or product, it was possible to monitor the relative quantity of reactants or products during a catalytic reaction.

Catalysis↗
PubMedDate not supplied

High-throughput gas sensing screening of surface-doped In(2)O(3).

The effect of various surface doping elements on the electrical and gas sensing properties of indium(III) oxide thick films sensors was investigated by means of high-throughput impedance spectroscopy (HTIS). Some doping elements lead to changes in both the conductivity in air as well as in the gas sensing properties towards oxidizing (NO(2), NO) and reducing (H(2), CO, propene) gases. Correlations between the sensing and the electrical properties in reference atmosphere indicate that the effect of the doping elements can be ascribed to an influence on the oxidation state of the metal oxide surface rather that to an interaction with the respective testing gases. An equation for the description of the temperature-dependent conductivity in air and nitrogen is proposed to describe the oxidation state of the metal oxide surface taking into account sorption of oxygen. Furthermore, a model associating the sensing properties and the oxidation state of the surface layer of the metal oxide based on oxygen spillover from doping element particles to the metal oxide surface is introduced.

Combinatorial Chemistry Techniques↗
PubMed2004

Genome annotation by high-throughput 5' RNA end determination.

Complete gene identification and annotation, including alternative transcripts, remains a challenge in understanding genome organization. Such annotation can be achieved by a combination of computational analysis and experimental confirmation. Here, we describe a high-throughput technique, trans-spliced exon coupled RNA end determination (TEC-RED), that identifies 5' ends of expressed genes in nematodes. TEC-RED can distinguish coding regions from regulatory regions and identify genes as well as their alternative transcripts that have different 5' ends. Application of TEC-RED to approximately 10% of the Caenorhabditis elegans genome yielded tags 75% of which experimentally verified predicted 5'-RNA ends and 25% of which provided previously unknown information about 5'-RNA ends, including the identification of 99 previously unknown genes and 32 previously unknown operons. This technique will be applicable in any organisms that have a trans-splicing reaction from spliced leader RNA. We also describe an efficient sequential method for concatenating short sequence tags for any serial analysis of gene expression-like techniques.

Animals↗
PubMed2005

High-throughput mapping of a dynamic signaling network in mammalian cells.

Signaling pathways transmit information through protein interaction networks that are dynamically regulated by complex extracellular cues. We developed LUMIER (for luminescence-based mammalian interactome mapping), an automated high-throughput technology, to map protein-protein interaction networks systematically in mammalian cells and applied it to the transforming growth factor-beta (TGFbeta) pathway. Analysis using self-organizing maps and k-means clustering identified links of the TGFbeta pathway to the p21-activated kinase (PAK) network, to the polarity complex, and to Occludin, a structural component of tight junctions. We show that Occludin regulates TGFbeta type I receptor localization for efficient TGFbeta-dependent dissolution of tight junctions during epithelial-to-mesenchymal transitions.

Activin Receptors, Type I↗
PubMed2005

Development of a simple assay protocol for high-throughput screening of Mycobacterium tuberculosis glutamine synthetase for the identification of novel inhibitors.

Mycobacterium tuberculosis glutamine synthetase (GS) is an essential enzyme involved in the pathogenicity of the organism. The screening of a compound library using a robust high-throughput screening (HTS) assay is currently thought to be the most efficient way of getting lead molecules, which are potent inhibitors for this enzyme. The authors have purified the enzyme to a >90% level from the recombinant Escherichia coli strain YMC21E, and it was used for partial characterization as well as standardization experiments. The results indicated that the Km of the enzyme for L-glutamine and hydroxylamine were 60 mM and 8.3 mM, respectively. The Km for ADP, arsenate, and Mn2+ were 2 microM, 5 microM, and 25 microM, respectively. When the components were adjusted according to their Km values, the activity remained constant for at least 3 h at both 25 degrees C and 37 degrees C. The Z' factor determined in microplate format indicated robustness of the assay. When the signal/noise ratios were determined for different assay volumes, it was observed that the 200-microl volume was found to be optimum. The DMSO tolerance of the enzyme was checked up to 10%, with minimal inhibition. The IC50 value determined for L-methionine S-sulfoximine on the enzyme activity was 3 mM. Approximately 18,000 small molecules could be screened per day using this protocol by a Beckman Coulter HTS setup.

Dimethyl Sulfoxide↗
PubMed2006

Multiplexing Fluo-4 NW and a GeneBLAzer transcriptional assay for high-throughput screening of G-protein-coupled receptors.

Activation of G-protein-coupled receptors (GPCRs) leads to a cascade of signaling events, including calcium mobilization and downstream transcriptional activation of various proteins. Two commonly used methods of high-throughput screening for GPCRs include calcium-sensitive dyes, such as Fluo-4 NW, and reporter gene assays, such as beta-lactamase. To determine whether the advantages of each assay format could be combined by multiplexing, Jurkat and CHO-K1 cell lines over-expressing the M1 muscarinic receptor and beta-lactamase under control of an NFAT response element were tested in a multiplexed format. The Jurkat cell line was further screened with a subset of the LOPAC(1280) library. The multiplexing assay was compatible with both the CHO-K1 and Jurkat cell lines. For the screen, there was 100% correlation of on-target hits in the multiplexed format, and several false positives with each assay format were identified. Therefore, not only can the assays be multiplexed, but by multiplexing, the false positives associated with each assay format also could be easily identified. In addition to enhanced reliability, this method saves time and money because only half the amount of compounds, cells, and consumables are needed to screen a cell line in a multiplexed mode versus separate screening by both methods.

Aniline Compounds↗
PubMed2006

Development of high-throughput spermidine synthase activity assay using homogeneous time-resolved fluorescence.

Spermidine synthase (SPDS) catalyzes transfer of the propylamine group from decarboxylated S-adenosylmethionine (dcSAM) to putrescine to yield methylthioadenosine (MTA) and spermidine. SPDS plays a regulatory role in cell proliferation and differentiation. This article describes the development of a high-throughput SPDS activity assay using homogeneous time-resolved fluorescence (HTRF) based on energy transfer from europium cryptate as a donor to crosslinked allophycocyanin (XL665) as an acceptor. First a highly specific anti-MTA monoclonal antibody, MTA-7H8, was generated, and then a competitive immunoassay for MTA determination was developed using europium cryptate-labeled MTA-7H8 and XL665-labeled MTA. In our homogeneous immunoassay, the percentage molar cross-reactivity of dcSAM with MTA-7H8 was 0.01% and the detection limit of MTA was 2.6 pmol/well. Our HTRF assay uses only one assay plate in which both enzyme reaction and MTA determination can be done successively. Therefore, our method can enable automatic screening of SPDS inhibitors from large numbers of samples.

Antibodies, Monoclonal↗
PubMed2006

'Information-Based-Acquisition' (IBA) technique with an ion-trap/time-of-flight mass spectrometer for high-throughput and reliable protein profiling.

Highly complex protein mixtures can be analyzed after proteolysis using liquid chromatography/mass spectrometry (LC/MS). In an LC/MS run, intense peptide ions originating from high-abundance proteins are preferentially analyzed using tandem mass spectrometry (MS(2)), so obtaining the MS(2) spectra of peptide ions from low-abundance proteins is difficult even if such ions are detected. Furthermore, the MS(2) spectra may produce insufficient information to identify the peptides or proteins. To solve these problems, we have developed a real-time optimization technique for MS(2), called the Information-Based-Acquisition (IBA) system. In a preliminary LC/MS run, a few of the most intense ions detected in every MS spectrum are selected as precursors for MS(2) and their masses, charge states and retention times are automatically registered in an internal database. In the next run, a sample similar to that used in the first run is analyzed using database searching. Then, the ions registered in the database are excluded from the precursor ion selection to avoid duplicate MS(2) analyses. Furthermore, real-time de novo sequencing is performed just after obtaining the MS(2) spectrum, and an MS(3) spectrum is obtained for accurate peptide identification when the number of interpreted amino acids in the MS(2) spectrum is less than five. We applied the IBA system to a yeast cell lysate which is a typical crude sample, using a nanoLC/ion-trap time-of flight (IT/TOF) mass spectrometer, repeating the same LC/MS run five times. The obtained MS(2) and MS(3) spectra were analyzed by applying the Mascot (Matrix Science, Boston, MA, USA) search engine to identify proteins from the sequence database. The total number of identified proteins in five LC/MS runs was three times higher than that in the first run and the ion scores for peptide identification also significantly increased, by about 70%, when the MS(3) spectra were used, combined with the MS(2) spectra, before being subjected to Mascot analysis.

Chromatography, High Pressure Liquid↗
PubMed2001

High-throughput genetic analysis using time-resolved fluorometry and closed-tube detection.

Robust methods for genetic analysis are required for efficient exploitation of the constantly accumulating genetic information. We describe a closed-tube genotyping method suitable for high-throughput screening of genetic markers. The method is based on allele-specific probes labeled with an environment-sensitive lanthanide chelate, the fluorescence intensity of which is significantly increased upon PCR amplification of a complementary target. Genomic DNA samples were analyzed in an insulin gene single nucleotide polymorphism (SNP) assay using universal amplification primers and probes that recognized the two different alleles. The feasibility of dry reagent based all-in-one PCR assays was tested using another diabetes-related genetic marker, human leukocyte antigen DQB1 allele *0302 as a model analyte in a dual-color, closed-tube end-point assay. There was a 100% correlation between the novel SNP assay and a conventional PCR restriction fragment length polymorphism assay. It was also demonstrated that using real-time monitoring, accurate genotyping results can be obtained despite strongly cross-reacting probes, minimizing the time and effort needed for optimization of probe sequence. Throughput can be maximized by using predried PCR mixtures that are stable for at least 6 months. This homogenous, all-in-one dry reagent assay chemistry permits cost-effective genetic screening on a large scale.

Alleles↗