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PubMed2004

High-throughput expression in microplate format in Saccharomyces cerevisiae.

We have developed a high-throughput technology that allows parallel expression, purification, and analysis of large numbers of cloned cDNAs in the yeast Saccharomyces cerevisiae. The technology is based on a vector for intracellular protein expression under control of the inducible CUP1 promoter, where the gene products are fused to specific peptide sequences. These N-terminal and C-terminal epitope tags allow the immunological identification and purification of the gene products independent of the protein produced. By introducing the method of recombinational cloning we avoid time-consuming re-cloning steps and enable the easy switching between different expression vectors and host systems.

Carrier Proteins↗
PubMed1977

High-quality, high-throughput, early whole-body bone scanning.

99mTc-IDP allows for high-quality, high-throughput and early whole-body bone scanning. Whole-body bone scans obtained at 30 min after intravenous administration of the tracer were compared with the standard and delayed 180 min p.i images. A subtraction technique allowed for the anlysis of the 30 min scans and it was found that in both types of investigation the same clinical information was present. This procedure will therefore increase considerably the throughput of whole-body bone scans in a busy department of nuclear medicine. It is however inadequate for detailed analysis of the skeleton (evaluation of benign bone disease).

Adult↗
PubMed1993

A reliable high-performance liquid chromatography assay for high-throughput routine cyclosporin A monitoring in whole blood.

We report here a reliable high-performance liquid chromatography-ultraviolet assay for routine assay of cyclosporin A (CsA) in whole blood using solid-phase extraction. This assay is linear, between 20 and 2,000 micrograms/L, with correlation coefficients > 0.998 for five consecutive standard curves. All coefficients of variation (CV) were < 8% at CsA concentrations of 45, 480, and 1,800 micrograms/L, with the exception of the between-day CV at 45 micrograms/L, which was < 15%. The relative accuracy of the method is > 94% at 45, 480, and 1,800 micrograms/L. The mean recoveries for CsA and cyclosporin D (internal standard) were 38.2 +/- 4.8% (n = 45) and 40.1 +/- 6.7% (n = 45), respectively. This method has proven to be reliable and robust in a high-throughput therapeutic drug monitoring laboratory.

Chemistry, Clinical↗
PubMed2000

Antibody arrays for high-throughput screening of antibody-antigen interactions.

We have developed a novel technique for high-throughput screening of recombinant antibodies, based on the creation of antibody arrays. Our method uses robotic picking and high-density gridding of bacteria containing antibody genes followed by filter-based enzyme-linked immunosorbent assay (ELISA) screening to identify clones that express binding antibody fragments. By eliminating the need for liquid handling, we can thereby screen up to 18,342 different antibody clones at a time and, because the clones are arrayed from master stocks, the same antibodies can be double spotted and screened simultaneously against 15 different antigens. We have used our technique in several different applications, including isolating antibodies against impure proteins and complex antigens, where several rounds of phage display often fail. Our results indicate that antibody arrays can be used to identify differentially expressed proteins.

Amino Acid Sequence↗
PubMedDate not supplied

Discovering reliable protein interactions from high-throughput experimental data using network topology.

OBJECTIVE: Current protein-protein interaction (PPI) detection via high-throughput experimental methods, such as yeast-two-hybrid has been reported to be highly erroneous, leading to potentially costly spurious discoveries. This work introduces a novel measure called IRAP, i.e. "interaction reliability by alternative path", for assessing the reliability of protein interactions based on the underlying topology of the PPI network. METHODS AND MATERIALS: A candidate PPI is considered to be reliable if it is involved in a closed loop in which the alternative path of interactions between the two interacting proteins is strong. We devise an algorithm called AlternativePathFinder to compute the IRAP value for each interaction in a complex PPI network. Validation of the IRAP as a measure for assessing the reliability of PPIs is performed with extensive experiments on yeast PPI data. All the data used in our experiments can be downloaded from our supplementary data web site at . RESULTS: Results show consistently that IRAP measure is an effective way for discovering reliable PPIs in large datasets of error-prone experimentally-derived PPIs. Results also indicate that IRAP is better than IG2, and markedly better than the more simplistic IG1 measure. CONCLUSION: Experimental results demonstrate that a global, system-wide approach-such as IRAP that considers the entire interaction network instead of merely local neighbors-is a much more promising approach for assessing the reliability of PPIs.

Algorithms↗
PubMed2006

Development of a high-throughput screen for soluble epoxide hydrolase inhibition.

Mammalian soluble epoxide hydrolase (sEH) represents a highly promising new target for drug development. Chemical inhibition of this enzyme in animal models was shown to treat hypertension and vascular inflammation as well as related syndromes. Existing sEH inhibitors are relatively potent and specific. However, the low solubility and relatively fast metabolism of described sEH inhibitors make them less than therapeutically efficient, stating the need for novel inhibitor structures. Therefore, a series of alpha-cyanoester and alpha-cyanocarbonate epoxides were evaluated as potential human sEH (HsEH) substrates for the high-throughput screen (HTS) of compound libraries. (3-Phenyl-oxiranyl)-acetic acid cyano-(6-methoxy-naphthalen-2-yl)-methyl ester (PHOME), which displayed the highest aqueous stability and solubility, was selected for the development of an HTS assay with long incubation times at room temperature. Concentrations of HsEH and PHOME were optimized to ensure assay sensitivity, reliability, and reproducibility. Assay validation, which employed these optimized concentrations, resulted in good accuracy (60-100%) and high precision (<7% relative standard deviation). In addition, an overall Z' value of 0.7 proved the system's robustness and potential for HTS. The developed assay system will be a valuable tool to discover new structures for the therapeutic inhibition of sEH to treat various cardiovascular diseases.

Enzyme Inhibitors↗
PubMed2003

A high-throughput on-line microdialysis-capillary assay for D-serine.

A high-throughput method is described for the analysis of D-serine and other neurotransmitters in tissue homogenates. Analysis is performed by microdialysis-capillary electrophoresis (CE) with laser-induced fluorescence (LIF) detection in a sheath flow detection cell. Sample pretreatment is not required as microdialysis sampling excludes proteins and cell fragments. Primary amines are derivatized on-line with o-phthaldialdehyde (OPA) in the presence of beta-mercaptoethanol followed by on-line CE-LIF analysis. Under the separation conditions described here, D-serine is resolved from L-serine and other primary amines commonly found in biological samples. Each separation requires less than 22 s. Eliminating the need for sample pretreatment and performing the high-speed CE analysis on-line significantly reduces the time required for D-serine analysis when compared with traditional methods. This method has been used to quantify D-serine levels in larval tiger salamander retinal homogenates, as well as dopamine, gamma-amino-n-butyric acid (GABA), glutamate and L-aspartate. D-serine release from an intact retina was also detected.

Animals↗
PubMed2006

Microplate orbital mixing improves high-throughput cell-based reporter assay readouts.

Reporter assays are commonly used for high-throughput cell-based screening of compounds, cDNAs, and siRNAs due to robust signal, ease of miniaturization, and simple detection and analysis. Among the most widely used reporter genes is the bioluminescent enzyme luciferase, which, when exposed to its substrate luciferin upon cell lysis, yields linear signal over a dynamic range of several orders of magnitude. Commercially available luciferase assay formulations have been developed permitting homogeneous, single-step cell lysis and reporter activity measurements. Assay conditions employed with these formulations are typically designed to minimize well-to-well luminescence variability due to variability in dispensing, evaporation, and incomplete sample mixing. The authors demonstrate that incorporating a microplate orbital mixing step into 96- and 384-well microplate cell-based luciferase reporter assays can greatly improve reporter readouts. They have found that orbital mixing using commercially available mixers facilitates maximal luciferase signal generation from high cell density-containing samples while minimizing variability due to partial cell lysis, thereby improving assay precision. The authors fully expect that widespread availability of mixers with sufficiently small orbits and higher speed settings will permit gains in signal and precision in the 1536-well format as well.

Animals↗
PubMed2000

Commercial high speed machines open new opportunities in high throughput flow cytometry (HTFC).

Two recent events have opened a new domain of flow cytometry applications which we term high throughput flow cytometry (HTFC). The release of a commercial high speed sorter in 1994 placed HTFC within the reach of anyone who could buy one of the new machines and not just the handful of advanced laboratories worldwide that had custom built their own high speed sorters. The advent in 1999 of HTFC analysis capabilities of 100000 cells/s marks the second stage in this enabling of HTFC. We describe the technical basis of HTFC. The commercial high speed sorters measure cells in dead-times three to six times shorter than conventional machines. They can sort with high yield and high purity at rates from 25000 to 60000 cells/s, depending on their settings, mainly by virtue of their use of high drop creation rates 100000 drops/s or more. Finally, one series can analyse the measured cells at rates exceeding these sort-rates and at least six times faster than conventional sorters could. The performance of the systems made by the three manufacturers can be readily assessed for single laser systems. Comparison becomes difficult for multiple beam machines, due to requirements for multi-beam sampling for each cell and due to the demands of fluorescence compensation between signals from one laser and between signals from two or three lasers. Applications are described in the field of rare cell analysis and isolation as well as from sorting of abundant cell populations.

Animals↗
PubMed2005

Surface plasmon resonance imaging-based protein arrays for high-throughput screening of protein-protein interaction inhibitors.

The E7 protein produced by high-risk human papillomavirus (HPV) induces a degradation of the retinoblastoma tumor suppressor RB through direct interaction, which suggests that an inhibitor for the interaction can be a potential anticancer drug. A surface plasmon resonance (SPR) imaging-based protein array chip was developed for the high-throughput screening of inhibitor molecules targeting RB-E7 interaction. The glutathione S-transferase-fused E7 protein (GST-E7) was first layered onto a glutathionylated gold chip surface that had been designed to specifically bind to GST-fused proteins. Subsequently, a microarrayer was used to spot the hexa-histidine-tagged RB proteins (His(6)-RB) onto the GST-E7-layered gold chip surface, and the resulting SPR image was analyzed. Upon increased His(6)-RB concentration in the spotting solution, the SPR signal intensity increased proportionally, indicating that His(6)-RB bound to GST-E7 in a concentration-dependent manner. The His(6)-RB/GST-E7 interaction was challenged by spotting the His(6)-RB solution in the presence of a RB binding peptide (PepC) derived from a motif on E7. The SPR imaging data showed that PepC inhibited the His(6)-RB/GST-E7 interaction in a concentration-dependent manner. Our results show that the SPR imaging-based protein array chip can be applied to screen small molecule inhibitors that target protein-protein interaction.

Papillomavirus E7 Proteins↗
PubMed1996

Analysis of heregulin-induced ErbB2 phosphorylation with a high-throughput Kinase receptor activation enzyme-linked immunosorbant assay.

A rapid, sensitive, and high-throughput assay has been developed to quantify ligand-induced receptor tyrosine kinase activation in terms of receptor phosphorylation. The assay, termed a kinase receptor activation enzyme-linked immunosorbant assay (KIRA-ELISA), consists of two separate microtiter plates, one for cell culture, ligand stimulation, and cell lysis/receptor solubilization and the other plate for receptor capture and phosphotyrosine ELISA. The assay was developed for analysis of heregulin-induced ErbB2 activation and utilizes the stimulation of intact receptor on the adherent breast carcinoma cell line, MCF-7. Membrane proteins are solubilized via Triton X-100 lysis and the receptor is captured in ELISA wells coated with ErbB2-specific antibodies with no cross-reaction to ErbB3 or ErbB4. The degree of receptor phosphorylation is then quantified by antiphosphotyrosine ELISA. A reproducible standard curve is generated with a EC(50) of approximately 360 pM for heregulin beta 1(177-244) (HRG beta 1(177-244). When identical samples of HRG beta 1(177-244) are analyzed by both the KIRA-ELISA and quantitative antiphosphotyrosine Western blot analysis, the results correlate very closely with one another. The assay described in this report is able to specifically quantify tyrosine phosphorylation of ErbB2 that results from the interaction of HRG with ErbB3 and/or ErbB4.

Animals↗
PubMed1999

Antisense oligonucleotides: a systematic high-throughput approach to target validation and gene function determination.

Antisense technology provides a high-throughput and systematic approach to drug target validation and gene function discovery. In combination with other emerging technologies (such as microarrays), this technology will enable efficient evaluation of the sequence data generated by the Human Genome Project. The authors review recent advances in the antisense field and discuss the potential use of antisense technology for functional genomics.

Journal Article↗
PubMed2001

High-throughput microfabricated CE/ESI-MS: automated sampling from a microwell plate.

A new design for high-throughput microfabricated capillary electrophoresis/electrospray mass spectrometry (CE/ ESI-MS) with automated sampling from a microwell plate is presented. The approach combines a sample-loading port, a separation channel, and a liquid junction, the latter for coupling the device to the MS with a miniaturized subatmospheric electrospray interface. The microdevice was attached to a polycarbonate manifold with external electrode reservoirs equipped for electrokinetic and pressure-fluid control. A computer-activated electropneumatic distributor was used for both sample loading from the microwell plate and washing of channels after each run. Removal of the electrodes and sample reservoirs from the microdevice structure significantly simplified the chip design and eliminated the need both for drilling access holes and for sample/buffer reservoirs. The external manifold also allowed the use of relatively large reservoirs that are necessary for extended time operation of the system. Initial results using this microfabricated system for the automated CE/ESI-MS analysis of peptides and protein digests are presented.

Amino Acid Sequence↗
PubMedDate not supplied

Realities of high-throughput liquid chromatography/mass spectrometry purification of large combinatorial libraries: a report on overall sample throughput using parallel purification.

We report on the development of a validated, streamlined high-throughput process for the purification of parallel-synthesis-derived combinatorial libraries. The steps involved in this library purification process include dissolution of dry films of crude synthetic material, dual-column LC/MS purification, dual-column postpurification analysis, quantitation, reformatting, and submission of pure compounds for registration. Although the purification and postpurification analysis times decreased essentially linearly as a function of the number of HPLC columns employed, it was not possible to decrease the total purification process time linearly as a function of the number of columns employed in the system. This was due primarily to the fact that numerous steps in the total purification process are independent of sample analysis and purification (e.g., evaporation, reconstitution, and reformatting, etc.). Additionally, experiments were also performed to assess whether separate gradient pumps were necessary for each channel of this two-channel LC/MS or if acceptable results could be reliably obtained by splitting the flow from one set of gradient pumps between two HPLC columns. On the basis of the parallel, two-column LC/MS system employed in this work, throughput estimates were extrapolated to more massively parallel systems (e.g., four-channel LC/MS).

Amino Acids↗
PubMedDate not supplied

High-throughput purification of combinatorial arrays.

Performance and reproducibility of the Biotage Parallex, a high-throughput purification system, was evaluated using known standards. The results indicate that parallel purification is a robust technique for purifying large numbers of compounds. Results from one of the first libraries to be purified on the Biotage Parallex are presented and discussed. Since fractionation by UV can often result in a large number of fractions, threshold trigger versus yield and number of fractions was also investigated. This approach was used to purify an array of 4320 compounds, produced by an 11-step solid-phase synthesis in Irori MicroKans. Ninety-three percent of the compounds were successfully processed, with >90% having purity >95%.

Journal Article↗
PubMedDate not supplied

Design and prioritization of plates for high-throughput screening.

A general algorithm for the prioritization and selection of plates for high-throughput screening is presented. The method uses a simulated annealing algorithm to search through the space of plate combinations for the one that maximizes some user-defined objective function. The algorithm is robust and convergent, and permits the simultaneous optimization of multiple design objectives, including molecular diversity, similarity to known actives, predicted activity or binding affinity, and many others. It is shown that the arrangement of compounds among the plates may have important consequences on the ability to design a well-targeted and cost-effective experiment. To that end, two simple and effective schemes for the construction of homogeneous and heterogeneous plates are outlined, using a novel similarity sorting algorithm based on one-dimensional nonlinear mapping.

Algorithms↗
PubMed2002

A high-throughput optical screening method for the optimization of colloidal water oxidation catalysts.

A high-throughput method has been developed for screening and optimization of colloidal water oxidation catalysts. The catalysts are irradiated in parallel by visible light from an overhead projector in solutions containing tris(2,2'-bipyridyl)ruthenium(II) (Ru(bpy)(3)(2+)) and persulfate. The array of reaction solutions is held in a 96-well plate, and absorbance readings are taken intermittently using a bioassay plate reader. The absorbance at 430 nm is indicative of the amount of Ru(bpy)(3)(2+) remaining in solution. The best catalysts give the most persistent absorbance, because the oxygen evolution reaction is kinetically competitive with decomposition of Ru(bpy)(3)(3+). Reagent concentrations were varied using a factorial design-of-experiment approach in order to optimize reaction conditions for a IrO(2).xH(2)O colloidal catalyst. A higher colloid concentration, a lower Ru(bpy)(3)(2+) concentration, and a higher pH buffer doubled the number of turnovers relative to the original conditions. Metal oxide colloids consisting of IrO(2).xH(2)O doped with varying amounts of Pt, Ru, and Os were made using a parallel microwave synthesis technique and were tested both by the parallel screening method and by direct measurement of oxygen evolution. The correlation between the two methods was good, with Ir-Pt-Os oxide compositions showing the highest activity. The effect of adding small amounts of Pt and Os to IrO(2).xH(2)O appears to be predominantly to reduce the particle size of the colloids.

Journal Article↗