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At least 199 records · Page 11Linked to original sources
PubMed1998

Development of a scintillation proximity assay for high-throughput measurement of intact parathyroid hormone.

A simple, high-throughput scintillation proximity assay (SPA) for parathyroid hormone (1-84) (PTH) has been developed. Fifteen commercially available N-terminal and six C-terminal anti-PTH antibodies were evaluated for detection of human PTH(1-84). Two C-terminal antibodies (CR1073M and 10-P55) gave the most consistent results. Using one of these antibodies (10-P55), an assay was developed with a sensitivity of 4 pg/ml for human and rat PTH(1-84). Porcine PTH(1-84) was not detectable. The intra-assay and inter-assay coefficients of variation for a 467 pg/ml sample were 6. 1 and 6.5%, respectively, and for a 21 pg/ml sample, 6.2 and 4.4%. Human PTH(1-34), while not detected in the assay, interfered with the detection of PTH(1-84). Smaller fragments [for example, human PTH(3-34)] and a C-terminal PTH fragment [PTH(53-84)] did not interfere in the assay. The procedure gave 106-110% recovery of human PTH(1-84) spiked into samples. Immunoreactive PTH concentrations in serum of rats administered EGTA were determined by SPA and by a commercially available PTH immunoassay. There was a good correlation between the two assays with significant increases in serum immunoreactive PTH concentrations at 15 and 30 min after EGTA injection and a rapid decrease to baseline values by 60 min. The SPA gives a high-throughput method for simply and accurately determining PTH(1-84) concentrations in serum.

Animals↗
PubMed2004

Blood coagulation kinetics: high throughput method for real-time reaction monitoring.

A high throughput 384-well plate assay of blood function in 60 microl reactions with the fluorogenic thrombin substrate, boc-VPR-MCA, allowed for real-time monitoring of coagulation under a diverse set of reaction conditions. Using recalcified, citrated whole blood diluted 3-fold with corn trypsin inhibitor (to block Factor XIIa), addition of 0 to 13.8 pM of tissue factor (TF) reduced the time of maximal rate of thrombin production T(max) from 45 min to 11 min. Over this range of TF,T(max) was reduced from 35 min to 6 min by co-addition of 10 nM convulxin to activate platelets via GPVI. The maximal rate of thrombin production at T(max) was not a function of exogenously-added TF,Va, or reVIIa, but increased 30% with added convulxin. Addition of 0.07 to 0.7 pM TF along with convulxin produced small, but detectable reductions in T(max). Addition of up to 0.67 nM reVIIa reduced T(max) by up to 53% in the range of 0.7 to 7 pM TF. Interestingly, platelet factor 4 (2.7 microM) caused a prolongation of T(max) from 45 min to 78 min at 0 TF, while protamine (1.8 microM) reduced T(max) to 30 min at 0 TF. Finally, combinatorial reaction studies with exogenously-added ADP, histamine, fMLP, indomethacin, anti-CD18, and fibrinogen revealed no unusual synergies amongst the agents, but demonstrated a striking procoagulant activity of added fibrinogen, due to protease contaminants in the "purified" fibrinogen. This high throughput approach allowed automated profiling of blood (50 reactions/ml of blood) to generate large data sets for testing cellular-proteomic kinetic models, screening drug interactions, and potentially monitoring subtle changes in the functional phenotype of a patient blood sample.

Automation↗
PubMed2000

High-throughput protein crystallization.

The combinatorial chemistry industry has made major advances in the handling and mixing of small volumes, and in the development of robust liquid-handling systems. In addition, developments have been made in the area of material handling for the high-throughput drug screening and combinatorial chemistry fields. Lastly, improvements in beamline optics at synchrotron sources have enabled the use of flash-frozen micron-sized (10-50 microm) crystals. The combination of these and other recent advances will make high-throughput protein crystallography possible. Further advances in high-throughput methods of protein crystallography will require application of the above developments and the accumulation of success/failure data in a more systematic manner. Major changes in crystallography technology will emerge based on the data collected by first-generation high-throughput systems.

Automation↗
PubMed1999

Confocal fluorescence coincidence analysis: an approach to ultra high-throughput screening.

Fluorescence-based assay technologies play an increasing role in high-throughput screening. They can be classified into different categories: fluorescence polarization, time-resolved fluorescence, fluorescence resonance energy transfer, and fluorescence correlation spectroscopy. In this work we present an alternative analytical technique for high-throughput screening, which we call confocal fluorescence coincidence analysis. Confocal fluorescence coincidence analysis extracts fluorescence fluctuations that occur coincidently in two different spectral ranges from a tiny observation volume of below 1 fl. This procedure makes it possible to monitor whether an association between molecular fragments that are labeled with different fluorophores is established or broken. Therefore, it provides access to the characterization of a variety of cleavage and ligation reactions in biochemistry. Confocal fluorescence coincidence analysis is a very sensitive and ultrafast technique with readout times of 100 ms and below. This feature is demonstrated by means of a homogeneous assay for restriction endonuclease EcoRI. The presented achievements break ground for throughput rates as high as 10(6) samples per day with using only small amounts of sample substance and therefore constitute a solid base for screening applications in drug discovery and evolutionary biotechnology.

Base Sequence↗
PubMed2002

Extractionless and sensitive method for high-throughput quantitation of cetirizine in human plasma samples by liquid chromatography-tandem mass spectrometry.

Following a single 10-mg oral dose of cetirizine dihydrochloride to 24 healthy volunteers, the analyte was quantified in human plasma. Protein precipitation using acetonitrile (ACN) was followed by reversed-phase liquid chromatography and tandem mass spectrometry. The MS/MS method was optimised using a PE Sciex API 2000 triple quadrupole mass spectrometer in selected reaction monitoring (SRM) mode, using electrospray with positive ionisation. Oxybutynin was used as the internal standard. The assay method represents a robust, high-throughput, highly specific and sensitive quantitative assay procedure, with 0.5 ng/ml being the lowest plasma concentration that could be reliably quantified. The procedure involves minimal sample preparation, and is well suited to clinical studies of the drug involving large numbers of generated samples. Pre-dose as well as post-dose samples up to and including 48 h were quantified, and the data generated were used to determine the pharmacokinetic profile of the drug.

Cetirizine↗
PubMed2003

Development of a 7-day, 96-well Caco-2 permeability assay with high-throughput direct UV compound analysis.

PURPOSE: The aim was to replace the traditional 21-day Caco-2 permeability protocol by a more high-throughput assay. METHODS: Caco-2 cells were seeded at high density in 96-well plates in novel cell culture boxes. After 7 days, drug permeability studies were performed. Samples were analyzed by a new UV detection method. RESULTS: With increased cell seeding density. functional Caco-2 monolayers with polarized efflux transporters were established after 7 days in 96-well polycarbonate filter plates in standard medium. For faster feeding and to eliminate medium replacement in each individual well, plates were completely submerged in medium in novel cell culture boxes, and only medium outside the plate was exchanged. For high-throughput sample analysis, a novel UV-transparent transport buffer was established that allowed direct quantification of permeated drug from its UV absorption. In vitro permeability studies analyzing 22 passively absorbed drugs in the new model correlated well with reported human permeability values (r2 = 0.8725). CONCLUSIONS: The new 7-day. 96-well Caco-2 permeability model tight to UV analysis offers considerable time, cost, and resource savings compared to the traditional model. It has a potential for automation and makes it possible to determine the permeability of passively diffusing compounds and to classify them according to the BCS in a truly medium- to high-throughput mode.

Algorithms↗
PubMed2006

High-throughput Plasmodium falciparum growth assay for malaria drug discovery.

New therapeutic agents for the treatment of malaria, the world's most deadly parasitic disease, are urgently needed. Malaria afflicts 300 to 500 million people and results in 1 to 2 million deaths annually, and more than 85% of all malaria-related mortality involves young children and pregnant women in sub-Saharan Africa. The emergence of multidrug-resistant parasites, especially in Plasmodium falciparum, has eroded the efficacy of almost all currently available therapeutic agents. The discovery of new drugs, including drugs with novel cellular targets, could be accelerated with a whole-organism high-throughput screen (HTS) of structurally diverse small-molecule libraries. The standard whole-organism screen is based on incorporation of [3H]hypoxanthine and has liabilities, such as limited throughput, high cost, multiple labor-intensive steps, and disposal of radioactive waste. Recently, screens have been reported that do not use radioactive incorporation, but their reporter signal is not robust enough for HTS. We report a P. falciparum growth assay that is technically simple, robust, and compatible with the automation necessary for HTS. The assay monitors DNA content by addition of the fluorescent dye 4',6-diamidino-2-phenylindole (DAPI) as a reporter of blood-stage parasite growth. This DAPI P. falciparum growth assay was used to measure the 50% inhibitory concentrations (IC50s) of a diverse set of known antimalarials. The resultant IC50s compared favorably with those obtained in the [3H]hypoxanthine incorporation assay. Over 79,000 small molecules have been tested for antiplasmodial activity using the DAPI P. falciparum growth assay, and 181 small molecules were identified as highly active against multidrug-resistant parasites.

Animals↗
PubMed2025

Enzymes in high-throughput RNA sequencing: Applications and challenges.

High-throughput RNA sequencing provides genome-wide information on the dynamics of RNA in each cell and how the dynamics responds to environmental changes. Next-generation sequencing by the Illumina platform currently provides the highest information output as compared to other platforms. A key component of next generation sequencing of each RNA is the successful end-to-end reverse-transcription into a cDNA strand. This can be highly challenging given the propensity of each RNA to adopt ordered structures and to contain post-transcriptional modifications. While many reverse transcriptase (RT) enzymes have been developed over the years to maximize read-through of an RNA, their processivity and efficiency varies, raising the question of how to select the RT for the experiment at hand. Here, we use tRNA as a model for genome-wide sequencing, as tRNA has a stable secondary and tertiary structure and has a high density and wide variety of post-transcriptional modifications, presenting one of the most challenging problems of sequencing RNA. We compare the efficiency of end-to-end cDNA synthesis of tRNA among several recent RT enzymes and provide a general sequencing workflow that is applicable to most of these enzymes.

High-Throughput Nucleotide Sequencing↗
PubMed2005

High-throughput real-time RT-PCR assay to detect the exotic Newcastle Disease Virus during the California 2002--2003 outbreak.

During the 2002--2003 Exotic Newcastle Disease (END) outbreak in Southern California, a high-throughput real-time reverse transcriptase-polymerase chain reaction (RRT-PCR) system was developed to respond to the large diagnostic and surveillance sample workload. A 96-well RNA extraction method, using magnetic bead technology, combined with a 96-well RRT-PCR assay, allowed 1 technician to process and test more than 400 samples per day. A 3-technician team could complete testing on approximately 1,900 samples per day. The diagnostic sensitivity of the high-throughput RRT-PCR assay was 0.9967 (95% CI 0.9937-0.9997) based on 926 virus isolation confirmed positive samples. Diagnostic specificity using an initial 434 virus isolation confirmed negative samples was 100%. A diagnostic specificity of 0.9999 (95% CI 0.9999, >0.9999) was subsequently calculated on the basis of 2 false-positive results among 65,343 surveillance samples collected after the final END-positive case was confirmed in May 2003. Assay performance over 500 replicates, including reproducibility of the combined extraction and RRT-PCR amplification steps yielded a standard deviation of 0.70 RRT-PCR cycle thresholds (Ct) and a standard deviation of 0.59 Ct for the RRT-PCR steps alone. The high-throughput RRT-PCR developed for END contributed significantly to the 2002--2003 END control effort, reducing the predicted timeline for eradication from 3 years to just 11 months, primarily because of the large number of samples that could be rapidly tested. The 96-well approach described for high-throughput END RRT-PCR could be adapted to other rapid, high-volume testing needs, as required for potential foreign animal disease responses or intensive surveillance efforts.

Animals↗
PubMed2000

Development of multichannel devices with an array of electrospray tips for high-throughput mass spectrometry.

The basic principles of multichannel devices with an array of electrospray tips for high-throughput infusion electrospray ionization mass spectrometry (ESI-MS) have been developed. The prototype plastic devices were fabricated by casting from a solvent-resistant resin. The sample wells on the device were arranged in the format of the standard 96-microtiter well plate, with each sample well connected to an independent electrospray exit port via a microchannel with imbedded electrode. A second plastic plate with distribution microchannels was employed as a cover plate and pressure distributor. Nitrogen gas was used to pressurize individual wells for transport of sample into the electrospray exit port. The design of independent microchannels and electrospray exit ports allowed very high throughput and duty cycle, as well as elimination of any potential sample carryover. The device was placed on a computer-controlled translation stage for precise positioning of the electrospray exit ports in front of the mass spectrometer sampling orifice. High-throughput ESI-MS was demonstrated by analyzing 96 peptide samples in 480 s, corresponding to a potential throughput of 720 samples/h. As a model application, the device was used for the MS determination of inhibition constants of several inhibitors of HIV-1 protease.

Angiotensins↗
PubMed2001

High resolution, high-throughput amide deuterium exchange-mass spectrometry (DXMS) determination of protein binding site structure and dynamics: utility in pharmaceutical design.

Mass spectrometry-based peptide amide deuterium exchange techniques have proven to be increasingly powerful tools with which protein structure and function can be studied, and are unparalleled in their ability to probe sub-molecular protein dynamics. Despite this promise, the methodology has remained labor-intensive and time consuming, with substantial limitations in comprehensiveness (the extent to which target protein sequence is covered with measurable peptide fragments) and resolution (the degree to which exchange measurements can be ascribed to particular amides). I have developed and integrated a number of improvements to these methodologies into an automated high throughput, high resolution system termed Deuterium Exchange Mass Spectrometry (DXMS). With DXMS, complete sequence coverage and single-amide (amino acid) resolution are now rapidly accomplished. DXMS is designed to work well with large proteins and when only small amounts of material are available for study. Studies can be performed upon a receptor-ligand pair as they exist on or within a living cell (in vivo) without prior purification, allowing effective in situ study of integral membrane protein receptors. We have ambitious initiatives underway to make DXMS widely available both for basic academic research studies and commercial drug discovery efforts. In this paper I present an overview of DXMS technology and highlight some of the benefits it will provide in drug discovery and basic proteomics research.

Amides↗
PubMed2002

High-throughput logP measurement using parallel liquid chromatography/ultraviolet/mass spectrometry and sample-pooling.

A novel approach to high-throughput logP measurement based on liquid chromatography/ultraviolet/mass spectrometry (LC/UV/MS) is proposed. The logP value is determined by correlation with the logk value, where k is the capacity factor k = (t(r)-t(0))/t(0), with the logP value using a defined set of standards. Since the analyte retention time (t(r)) is determined from the appropriate extracted ion chromatogram (EIC), there are no interferences from impurities and this allows the pooling of multiple compounds into one injection. To ensure the accuracy and instrument robustness in a routine high-throughput environment, a simple and MS-friendly mobile phase consisting of 20 mM ammonium carbonate (pH 8.0) for basic compounds or 20 mM ammonium formate (pH 1.0) for acidic compounds, both in combination with methanol at a ratio of 45:55, is used. This approach has been successfully used on single as well as parallel multi-channel LC/UV/MS systems to screen small to large sets of lead compounds and their analogs. A high-throughput capability to analyze over 1000 compounds per day has been achieved.

Animals↗
PubMed2005

A live-cell high-throughput screening assay for identification of fatty acid uptake inhibitors.

We developed a live-cell high-throughput assay system using the baker's yeast Saccharomyces cerevisiae to screen for chemical compounds that will inhibit fatty acid uptake. The target for the inhibitors is a mammalian fatty acid transport protein (mmFATP2), which is involved in the fatty acid transport and activation pathway. The mmFATP2 was expressed in a S. cerevisiae mutant strain deficient in Fat1p-dependent fatty acid uptake and reduced in long-chain fatty acid activation, fat1Deltafaa1Delta. To detect fatty acid import, a fluorescent fatty acid analog, 4,4-difluoro-5-methyl-4-bora-3a,4a-diaza-s-indacene-3-dodecanoic acid (C1-BODIPY-C12), was incubated with cells expressing FATP2 in a 96-well plate. The mmFATP2-dependent C1-BODIPY-C12 uptake was monitored by measuring intracellular C1-BODIPY-C12 fluorescence on a microtiter plate reader, whereas extracellular fluorescence was quenched by a cell viability dye, trypan blue. Using this high-throughput screening method, we demonstrate that the uptake of the fluorescent fatty acid ligand was effectively competed by the natural fatty acid oleate. Inhibition of uptake was also demonstrated to occur when cells were pretreated with sodium azide or Triacsin C. This yeast live-cell-based assay is rapid to execute, inexpensive to implement, and has adequate sensitivity for high-throughput screening. The assay basis and limitations are discussed.

Biological Transport↗
PubMed2002

High throughput methods for gene cloning and expression.

We outline a high throughput process for the production of bacterial expression clones using automated liquid handlers. The protocol consists of a series of interlinked methods representing liquid manipulations or incubations on various stations of the automation system. The methods employ the ligation-independent cloning approach that enables the simultaneous production of plasmids for different expression systems. The current cloning protocol spans 3 days with a linear throughput of 400 targets per production run. This automated approach enables the production of large numbers of bacterial expression clones and ultimately purified proteins. Although they were developed for structural genomics, these molecular protocols can also be applied in high throughput strategies such as those used for site-specific mutagenesis or protein interaction studies.

Automation↗
PubMed2005

High-throughput solubility assay for purified recombinant protein immunogens.

A high-throughput assay is described for analysis of the solubility of purified recombinant proteins. The assay is based on affinity purification of proteins in the presence of chaotropic agents followed by a dilution and incubation step to investigate the solubility in the absence of high concentrations of such agents. The assay can be performed in a 96-well format, which makes it well suited for high-throughput applications. For 125 recombinant proteins expressed as part of an antibody-based proteomics effort, experimental solubility data were compared to calculated hydrophobicity values based on the amino acid sequence of each protein. This comparison showed only weak correlation between the theoretical and experimental values, which emphasizes the importance of experimental assays to determine the solubility of recombinant proteins.

Antibodies↗
PubMed1997

High-throughput RT-PCR analysis of multiple transcripts using a microplate RNA isolation procedure.

We have developed a high-throughput, multiplex reverse transcription PCR (RTPCR) assay that is suitable for the analysis of medium-to low-copy cellular RNA transcripts from small numbers of cells (10(4)). High throughput was attained by utilizing microplate-based RNA extraction and RTPCR protocols, followed by PCR product visualization of a multiwelled agarose gel, stained with SYBR Green I dye. The transcriptional assay was unaffected by solvents (dimethyl sulfoxide and methanol) routinely used in high-throughput drug screens at concentrations required for compound solubilization. Furthermore, it has been used successfully for the investigation of differential mRNA expression levels of tumor necrosis factor alpha (TNF-alpha) and Interleukin-1 beta (IL-1 beta) in lipopolysaccharide (LPS)-stimulated THP-1 cells (a human monocytic cell line) and the identification of specific IL-1 beta transcriptional inhibitors.

Benzothiazoles↗
PubMed2003

A high-throughput capillary assay for bacterial chemotaxis.

We present a high-throughput capillary assay in order to characterize the chemotactic response of the E. coli bacterium. We measure the number of organisms attracted into an array of 96 capillary tubes containing the attractant L-aspartate. The effect of bacterial concentration on the chemotactic response is reported. Such high-throughput assay can be used to characterize bacterial chemotaxis function of a wide range of biochemical parameters.

Chemotaxis↗
PubMedDate not supplied

High-throughput analysis in catalysis research using novel approaches to transmission infrared spectroscopy.

This study has demonstrated that high-throughput FTIR transmission measurements using a newly designed array-based support formed using silicon wells and a silicon wafer is a very useful and robust tool for the characterization of polymer composition for combinatorial materials research. The comonomer content in copolymers can be measured accurately with a fully automated throughput of >300 samples/day (8 h). The transmission measurement is more robust, reliable, and easier to automate than other spectroscopic methods. The support itself provides excellent resistance to aggressive organic solvents at elevated temperatures and allows the unattended deposition and preparation of polymer films for infrared analysis. Because of the excellent durability of the support with respect to the solvent, the support can be rinsed and reused many times. This high-throughput approach to infrared transmission spectroscopy can be used for measuring a wide array of polymer characteristics: vinyl content, geometrical isomers, crystallinity, and tacticity. As well, this IR approach can be used to predict the oxidative stability of the antioxidant packages. Because the support provides a means of containing hot polymer solutions while the solvent evaporates, the support is also suitable for high-throughput nanoindentation methods for the determination of modulus and other physical properties of the polymer.

Journal Article↗