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PubMed2006

High-throughput assays for DNA gyrase and other topoisomerases.

We have developed high-throughput microtitre plate-based assays for DNA gyrase and other DNA topoisomerases. These assays exploit the fact that negatively supercoiled plasmids form intermolecular triplexes more efficiently than when they are relaxed. Two assays are presented, one using capture of a plasmid containing a single triplex-forming sequence by an oligonucleotide tethered to the surface of a microtitre plate and subsequent detection by staining with a DNA-specific fluorescent dye. The other uses capture of a plasmid containing two triplex-forming sequences by an oligonucleotide tethered to the surface of a microtitre plate and subsequent detection by a second oligonucleotide that is radiolabelled. The assays are shown to be appropriate for assaying DNA supercoiling by Escherichia coli DNA gyrase and DNA relaxation by eukaryotic topoisomerases I and II, and E.coli topoisomerase IV. The assays are readily adaptable to other enzymes that change DNA supercoiling (e.g. restriction enzymes) and are suitable for use in a high-throughput format.

Clinical Enzyme Tests↗
PubMed1999

High Throughput Scintillation Proximity Assay for the Identification of FKBP-12 Ligands.

A high throughput scintillation proximity assay (SPA) was developed to identify novel ligands of FKBP-12, an immunophilin with peptidyl prolyl isomerase (rotamase) activity. Recombinant histidine-tagged FKBP-12 was expressed in Escherichia coli, purified by metal ion affinity chromatography, and immobilized to SPA beads by an antibody that recognizes the histidine tag of the recombinant protein. Using 1 nM [3H] FK506, a well-known macrolid ligand of FKBP-12, specific binding was saturable and accounted for 95% of total binding. Analysis of saturation and homologous displacement isotherms indicated the existence of a single binding site with a Kd value of 1.6 nM. The specificity of [3H] FK506 binding was demonstrated in displacement experiments and showed that rapamycin, another macrolid, was as active as FK506 (IC50 of 3.5 and 3.2 nM, respectively), whereas GPI-1046, a prototype of small molecular compounds with neurotrophic properties and affinity for FKBP-type immunophilins, was more than 1000-fold less active. The high signal-to-noise ratio of 30, together with small standard deviations, makes this novel assay well suited for automated high throughput screening.

Journal Article↗
PubMed2003

Synthetic DNA aptamers to detect protein molecular variants in a high-throughput fluorescence quenching assay.

Real-time protein detection in homogeneous solutions is necessary in many biotechnology and biomedical studies. The recent development of molecular aptamers, combined with fluorescence techniques, may provide an easy and efficient approach to protein elucidation. This report describes the development of a fluorescence-based assay with synthetic DNA aptamers that can detect and distinguish molecular variants of proteins in biological samples in a high-throughput process. We used an aptamer with high affinity for the B chain of platelet-derived growth factor (PDGF), labeled it with a fluorophore and a quencher at the two termini, and measured fluorescence quenching by PDGF. The specific quenching can be used to detect PDGF at picomolar concentrations even in the presence of serum and other cell-derived proteins in cell culture media. This is the first successful application of a synthetic aptamer for the detection of tumor-related proteins directly from the tumor cells. We also show that three highly related molecular variants of PDGF (AA, AB, and BB dimers) can be distinguished from one another in this single-step assay, which can be readily adapted to a microtiter plate assay for high-throughput analysis. The use of fluorescence quenching as a measure of binding between the DNA probe and the target protein eliminates potential false signals that may arise in traditional fluorescence enhancement assays as a result of degradation of the DNA aptamer by contaminating nucleases in biological specimens. This assay is applicable to proteins that are not naturally DNA binding. The excellent specificity, ultrahigh sensitivity, and simplicity of this one-step assay addresses a growing need for high-throughput methods that detect changes in the expression of gene products and their variants in cell cultures and biological specimens.

Animals↗
PubMed2001

HTPS flow cytometry: a novel platform for automated high throughput drug discovery and characterization.

The flow cytometer is unique among biomedical analysis instruments because it makes simultaneous and multiple optical measurements on individual cells or particles at high rates. High throughput flow cytometry represents a potentially important multifactorial approach for screening large combinatorial libraries of compounds. Limiting this approach has been the availability of instrumentation and methods in flow cytometry for automated sample handling on the scale required for drug discovery applications. Here, we describe an automated system in which a novel patented fluidics-based pharmacology platform, the HTPS (High Throughput Pharmacological System), is coupled to a flow cytometer using a recently described plug flow-coupling valve technology. Individual samples are aspirated sequentially from microplate wells and delivered to a flow cytometer for rapid multiparametric analysis. For primary screening to detect and quantify cell fluorescence in endpoint assays, a high-speed no-wash protocol enabled processing of 9-10 cell samples/min from 96-well microplates. In an alternate primary screening format, soluble receptor ligands were sampled from microplate wells at rates of 3-4 samples/minute and successfully assessed for the ability to elicit intracellular calcium responses. Experiments with fluorescent beads validated the accurate automated production by the HTPS of exponential and linear gradients of soluble compounds. This feature enabled rapid (2- to 3-min) characterization of the intracellular calcium dose response of myeloid cells to formyl peptide as well as the quantitative relationship between formyl peptide receptor occupancy and cell response. HTPS flow cytometry thus represents a powerful high throughput multifactorial approach to increase the efficiency with which novel bioresponse-modifying drugs may be identified and characterized.

Automation↗
PubMed2001

Recent developments in high-throughput mutation screening.

Screening of large sample materials for the presence of known or unknown mutations is a key element in pharmacogenomics. Although automated DNA sequencing has developed rapidly during the last decade, the technology is not well suited for projects involving analysis of hundreds of thousands of mutations. Consequently, a number of methods for high-throughput mutation screening have been developed. DNA microarrays and high-density oligonucleotide chips have proven to be well suited for parallel hybridisation-based analysis of hundreds or thousands of known mutations. Methods based on detection using matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI-TOF MS) have been developed. MALDI-TOF MS detection is limited to analysis of small DNA fragments but has a large potential for high-throughput single nucleotide polymorphism (SNP) analysis, due to a very fast analysis time and possibilities for automation. Currently, the best suited methods for high-throughput screening for unknown mutations are probably methods like single strand conformation polymorphism (SSCP) analysis or conformation sensitive gel electrophoresis (CSGE), combined with capillary array electrophoresis or denaturing high-performance liquid chromatography. This is due to a relatively short analysis time, potential for automation and a high sensitivity. The recent development of capillary array electrophoresis chips suggests that the analysis time for some of these methods may be reduced by one order of magnitude in the near future.

Chromatography, High Pressure Liquid↗
PubMed2006

Optimization of conditions for in vitro production of radical oxygen species and expression of tissue factor by canine mononuclear cells and granulocytes for use in high-throughput assays.

The purpose of this study was to optimize conditions for high throughput measurement of radical oxygen species (ROS) production and expression of tissue factor, also termed procoagulant activity, by canine leukocytes. Granulocytes and mononuclear cells were separated by density gradient centrifugation from peripheral blood collected on several occasions from three healthy large breed dogs. To determine optimal conditions for ROS production, granulocytes were incubated for 1 or 3h in PBG (PBS containing 0.5% BSA and 5mM glucose) or RPMI containing 10% fetal bovine serum (FBS); lipopolysaccharide (LPS), zymosan, peptidoglycan (PGN) and phorbol myristate acetate (PMA) were used as stimuli. ROS was assessed by conversion of the nonfluorescent dye dihydrorhodamine 123 to fluorescent rhodamine 123 by radical species released into the media. To identify optimal conditions for expression of tissue factor, mononuclear cells were incubated for 5h in RPMI containing different concentrations of heat-inactivated FBS (HI-FBS), and LPS, zymosan, PGN or PMA as stimuli. Expression of tissue factor was determined using a one-stage recalcification assay performed in an automated nephelometric coagulation analyzer. Neither LPS nor zymosan increased ROS production by granulocytes incubated in PBG media. In contrast, granulocytes incubated in RPMI had dose-dependent increases in ROS production in response to zymosan and PGN. ROS production was significantly increased by incubation with concentrations of LPS of 0.01microg/ml or greater, and by zymosan concentrations of 0.1microg/ml or greater. ROS production in response to incubation with PMA was significantly increased starting at 10(-7)M, and was significantly greater for cells incubated in RPMI than cells incubated in PBG. LPS-, zymosan- and PGN-stimulated procoagulant activity increased in a dose-dependent manner, whereas PMA-stimulated procoagulant activity peaked at 10(-7)M. Increasing concentrations of HI-FBS significantly increased LPS-, zymosan- and PGN-induced procoagulant activity of mononuclear cells. Results obtained in this study indicate production of ROS by canine granulocytes is optimal when these cells are incubated for 3h in RPMI with LPS (0.1microg/ml), zymosan (10 microg/ml), PGN (10 microg/ml), and PMA (10(-7)M). Furthermore, canine mononuclear cells express procoagulant activity in response to LPS, zymosan, PGN, and PMA, and responses to LPS, zymosan and PGN are enhanced by the addition of HI-FBS. These findings suggest that HI-FBS retains important serum proteins that facilitate interactions between each of these bacterial or yeast derived products and the mononuclear cells. Consequently, future studies regarding the regulation of procoagulant activity by canine mononuclear cells should be performed in the presence of HI-FBS. Both assays utilized in this study allow high throughput of samples, and therefore are appropriate choices for rapid screening of conditions and/or therapeutic interventions affecting the canine inflammatory system.

Animals↗
PubMed2004

Inkjet printing for high-throughput cell patterning.

The adaptation of inkjet printing technology to the complex fields of tissue engineering and biomaterial development presents the potential to increase progress in these emerging technologies through the implementation of this high-throughput capability via automated processes to enable precise control and repeatability. In this paper, a method of applying high-throughput inkjet printing to control cellular attachment and proliferation by precise, automated deposition of collagen is presented. The results indicate that commercial inkjet printing technology can be used to create viable cellular patterns with a resolution of 350 microm through the deposition of biologically active proteins. This method demonstrates a combination of off-the-shelf inkjet printing and biomaterials and has potential to be adapted to tissue engineering and colony patterning applications. Adapting this method into the three-dimensional construction of cellular structures for eventual high-throughput tissue engineering using a bottom-up approach is possible.

Animals↗
PubMedDate not supplied

A high-throughput search for electronic materials-thin-film dielectrics.

Parallel synthesis together with high-throughput screening was used to identify candidate materials for integrated circuit applications that demand a superior high permittivity dielectric thin film. Specifically, we developed a "continuous-composition spread" (CCS) technique to synthesize much of a pseudoternary oxide system in a single deposition and used this in conjunction with a high-throughput measurement protocol, thereby allowing each chemical system to be deposited and evaluated in about 24 h. This approach led to the identification of compositions in the Zr-Sn-Ti-O system with promising properties. The same technique was used to determine the optimum compositions as a function of processing parameters. Films with the composition Zr(.2)Sn(.2)Ti(.6)O(2) were then prepared using a conventional synthetic technique (on-axis sputtering) and were verified to have excellent properties. Thus, the CCS technique has demonstrated utility in rapidly identifying and developing a useful new material.

Combinatorial Chemistry Techniques↗
PubMed2003

High-throughput screening with immobilized metal ion affinity-based fluorescence polarization detection, a homogeneous assay for protein kinases.

Protein kinases are one of the most important target classes in high-throughput screening today. The use of generic assay technologies facilitates assay development for new targets and decreases the time needed for implementation of assays in robotic screening. For tyrosine kinases, several generic assay technology platforms are available. These technologies make use of high-affinity antibodies that discriminate between phosphorylated tyrosines and non-phosphorylated tyrosines. Similar generic antibodies specific for phosphoserine or phosphothreonine are lacking. Recently, a non-antibody-based fluorescence polarization assay for protein kinases has become available, called IMAP (Molecular Devices, Sunnyvale, CA). In this assay, a fluorescently labeled peptide substrate that is phosphorylated by kinase is captured on metal-derivatized nanoparticles. We have evaluated IMAP in high-throughput screening, and compared this technology with a competition fluorescence polarization immunoassay based on an antibody specific for a phosphorylated peptide substrate. A random collection of >250000 compounds was screened with the two assays. Fluorescent library compounds were identified by calculation of fluorescence intensity values from the screening data, and by assaying in the absence of fluorescent reagents. Fluorescence polarization artifacts were filtered out further by testing in an ELISA-based kinase assay. Our data show that IMAP is a robust technology for high-throughput screening of kinase targets, and suggest that it is less susceptible to fluorescence polarization artifacts than the competition fluorescence polarization immunoassay.

Artifacts↗
PubMed2004

High-throughput screening of novel peptide inhibitors of an integrin receptor from the hexapeptide library by using a protein microarray chip.

Protein microarray is an emerging technology that makes high-throughput analysis possible for protein-protein interactions and analysis of proteome and biomarkers in parallel. The authors investigated the application of a novel protein microarray chip, ProteoChip, in new drug discovery. Integrin alpha(v)beta(3) microarray immobilized on the ProteoChip was employed to screen new active peptides against the integrin from multiple hexapeptide sublibraries of a positional scanning synthetic peptide combinatorial library (PS-SPCL). The integrin alpha(v)beta(3)-vitronectin interaction was successfully demonstrated on the integrin microarray in a dose-dependent manner and was inhibited not only by the synthetic RGD peptide but also by various integrin antagonists on the integrin microarray chip. Novel peptide ligands with high affinity to the integrin were also identified from the peptide libraries with this chip-based screening system by a competitive inhibition assay in a simultaneous and high-throughput fashion. The authors have confirmed antiangiogenic functions of the novel peptides thus screened through an in vitro and in vivo angiogenesis assay. These results provide evidence that the ProteoChip is a promising tool for high-throughput screening of lead molecules in new drug development.

Cell Movement↗
PubMed2001

SwellGel: an affinity chromatography technology for high-capacity and high-throughput purification of recombinant-tagged proteins.

The revolution in genomics and proteomics is having a profound impact on drug discovery. Today's protein scientist demands a faster, easier, more reliable way to purify proteins. A high capacity, high-throughput new technology has been developed in Perbio Sciences for affinity protein purification. This technology utilizes selected chromatography media that are dehydrated to form uniform aggregates. The SwellGel aggregates will instantly rehydrate upon addition of the protein sample, allowing purification and direct performance of multiple assays in a variety of formats. SwellGel technology has greater stability and is easier to handle than standard wet chromatography resins. The microplate format of this technology provides high-capacity, high-throughput features, recovering milligram quantities of protein suitable for high-throughput screening or biophysical/structural studies. Data will be presented applying SwellGel technology to recombinant 6x His-tagged protein and glutathione-S-transferase (GST) fusion protein purification.

Binding Sites↗
PubMed2002

High-throughput HBV DNA and HCV RNA detection system using a nucleic acid purification robot and real-time detection PCR: its application to analysis of posttransfusion hepatitis.

BACKGROUND: A high-throughput detection system was developed for HBV DNA and HCV RNA. METHODS: A combination of real-time detection PCR using an automated system (PRISM 7700, PE Biosystems, Foster City, CA) and automatic viral nucleic acid extraction (BioRobot 9604, Qiagen, Hilden, Germany) was used as the high-throughput detection system. An internal control for HBV DNA detection was also developed. RESULTS: Testing of 96 samples for HBV and HCV was completed within 5 hours. The sensitivity of this system almost equals that of the manual method using nested PCR. The addition of an internal control for HBV detection did not affect the sensitivity of the method and confirmed the accuracy of results. It was possible to quantify HBV in HBV+ samples that contain more than 500 genome equivalents per mL. We started using this system from June 1999 for testing stored donor and patient samples to analyze cases of posttransfusion hepatitis and identified three HBV+ donations that were implicated in posttransfusion hepatitis B. CONCLUSION: The high-throughput detection system is a useful tool for HBV DNA and HCV RNA detection because it enables rapid and reliable testing of a large number of samples.

Automation↗
PubMed2004

Design of a compound screening collection for use in high throughput screening.

In this paper we introduce a quantitative model that relates chemical structural similarity to biological activity, and in particular to the activity of lead series of compounds in high-throughput assays. From this model we derive the optimal screening collection make up for a given fixed size of screening collection, and identify the conditions under which a diverse collection of compounds or a collection focusing on particular regions of chemical space are appropriate strategies. We derive from the model a diversity function that may be used to assess compounds for acquisition or libraries for combinatorial synthesis by their ability to complement an existing screening collection. The diversity function is linked directly through the model to the goal of more frequent discovery of lead series from high-throughput screening. We show how the model may also be used to derive relationships between collection size and probabilities of lead discovery in high-throughput screening, and to guide the judicious application of structural filters.

Chemistry, Pharmaceutical↗
PubMed2001

High-throughput generation and engineering of recombinant human antibodies.

The first version of the Human Combinatorial Antibody Library (HuCAL) is a single-chain Fv-based phage display library (HuCAL-scFv) with 2x10(9) members optimised for high-throughput generation and targeted engineering of human antibodies. 61% of the library genes code for functional scFv as judged by sequencing. We show here that since HuCAL-scFv antibodies are expressed in high levels in Escherichia coli, automated panning and screening in miniaturised settings (96- and 384-well format) have now become feasible. Additionally, the unique modular design of HuCAL-genes and -vectors allows the distinctly facilitated conversion of scFv into Fab, miniantibody and immunoglobulin formats, and the fusion with a variety of effector functions and tags not only convenient for therapeutic applications but also for high-throughput purification and detection. Thus, the HuCAL principle enables the rapid and high-throughput development of human antibodies by optimisation strategies proven useful in classical low molecular weight drug development. We demonstrate in this report that HuCAL is a very convenient source of human antibodies for various applications.

Animals↗
PubMed1999

A genetic strategy to eliminate self-activator baits prior to high-throughput yeast two-hybrid screens.

Large-scale sequencing projects have predicted high numbers of gene products for which no functional information is yet available. Hence, large-scale projects, such as gene knockouts, gene expression profiles, and protein-interaction mapping, are currently under way to initiate the understanding of the function of these gene products. The high-throughput strategies that are currently being developed to generate protein-interaction maps include automated versions of the yeast two-hybrid system. These strategies rely on the large-scale construction of DNA-binding domain/protein-of-interest hybrid constructs (DB-X baits). An inherent problem of large-scale two-hybrid systems is that a high percentage of cloned sequences encode polypeptides that, when fused to DB, can activate transcription in the absence of any two-hybrid-interacting partner protein. Here, we describe and validate a genetic strategy that efficiently eliminates such self-activator baits prior to screening procedures. The strategy is based on a negative-growth selection and is compatible with high-throughput settings.

DNA, Fungal↗
PubMedDate not supplied

Parallel synthesis and high throughput dissolution testing of biodegradable polyanhydride copolymers.

We have demonstrated that polycondensation reactions can be carried out in a combinatorial fashion and that the polymer library can be screened at high throughput using a rapid prototyping technique to fabricate multiwell substrates. A linearly varying compositional library of 100 different biodegradable polyanhydride random copolymers that are promising carriers for controlled drug delivery was designed, fabricated, and characterized by IR microscopy within a few hours. The polyanhydride copolymer library was based on 1,6-bis(p-carboxyphenoxy)hexane (CPH) and sebacic anhydride (SA) and was characterized with infrared microspectroscopy to determine the composition within each well. Since degradation and release rates depend on copolymer composition, we also developed new high-throughput methods to investigate drug release from this library of copolymers by designing specific wells for each task. A subset of this library was chosen, and a substrate was designed and fabricated to enable the synthesis and monitoring of dye dissolution from a range of polyanhydride copolymers in a parallel fashion using a CCD camera. Multisample substrates were fabricated with a novel rapid prototyping method that consists of an organic solvent-resistant array of 10 x 10 microwells of 2-muL volume each. The libraries were deposited with a custom-built liquid dispensing system consisting of a series of computer-controlled volume-dispensing pumps and XYZ motion stages. The parallel dye dissolution study displayed a decreasing rate of release with increasing CPH content. This result agrees with previously published data for dye release from poly(CPH-co-SA) copolymers. The methodology described in this work is amenable to numerous applications in the arenas of high-throughput polymer synthesis and characterization.

Biodegradation, Environmental↗
PubMedDate not supplied

Micro-X-ray fluorescence as a general high-throughput screening method for catalyst discovery and small molecule recognition.

A powerful high-throughput screening technique is described for the rapid screening of bead-based libraries for catalyst discovery and molecular recognition. Micro-X-ray fluorescence (MXRF) screens materials for elemental composition with mesoscale analysis. This method is nondestructive and requires minimal sample preparation and no special tags for analysis, and the screening time is dependent on the desired sensitivity. The speed, sensitivity, and simplicity of MXRF as a high-throughput screening technique were applied to screen bead-based libraries of oligopeptides for phosphate hydrolysis catalysts and molecular recognition of selective receptors for the degradation products and analogues of chemical warfare agents. This paper demonstrates the analytical or HTS capability of MXRF for combinatorial screening. It is meant only to show the capabilities of MXRF and is not meant as an exhaustive study of the catalyst and molecular recognition systems presented.

Catalysis↗
PubMed2002

Development of a high throughput screening assay for mitochondrial membrane potential in living cells.

The mitochondrion plays a pivotal role in energy metabolism in eukaryotic cells. The electrochemical potential across the mitochondrial inner membrane is regulated to cope with cellular energy needs and thus reflects the bioenergetic state of the cell. Traditional assays for mitochondrial membrane potential are not amenable to high-throughput drug screening. In this paper, I describe a high-throughput assay that measures the mitochondrial membrane potential of living cells in 96- or 384-well plates. Cells were first treated with test compounds and then with a fluorescent potentiometric probe, the cationic-lipophilic dye tetramethylrhodamine methyl ester (TMRM). The cells were then washed to remove free compounds and probe. The amount of TMRM retained in the mitochondria, which is proportional to the mitochondrial membrane potential, was measured on an LJL Analyst fluorescence reader. Under optimal conditions, the assay measured only the mitochondrial membrane potential. The chemical uncouplers carbonylcyanide m-chlorophenyl hydrazone and dinitrophenol decreased fluorescence intensity, with IC(50) values (concentration at 50% inhibition) similar to those reported in the literature. A Z' factor of greater than 0.5 suggests that this cell-based assay can be adapted for high-throughput screening of chemical libraries. This assay may be used in screens for drugs to treat metabolic disorders such as obesity and diabetes, as well as cancer and neurodegenerative diseases.

Animals↗