A telomerase enzymatic assay that does not use polymerase chain reaction, radioactivity, or electrophoresis

A telomerase assay has been developed for high-throughput screening in 96-well microtiter plates. A crude cell lysate which adds telomere repeats to a biotinylated DNA primer is the source of telomerase. The telomerase-extended primer is hybridized to a digoxigenin-labeled telomere antisense DNA probe. The hybrid is further processed by enzyme-linked immunosorbent assay (ELISA) as follows. The biotinylated hybrid is captured on streptavidin-coated microtiter plates. The immobilized hybrid is probed with alkaline phosphatase-antidigoxigenin and detected via chemiluminescent readout. The limit of detection of a chemically synthesized tetra-telomere repeat was about 10 attomoles. Apparent telomerase activity was detected in lysates of 293T cells. The signal to background for the assay (ratio of signal for the complete assay mixture divided by the signal for the assay mixture without primer) was around 10. An automated system that performed unattended runs of up to 17 96-well microtiter plates in 8h was constructed.     

Magnetic bead purification of M13 DNA sequencing templates.

A method for the preparation of multiple M13 DNA sequencing templates is described. No phenol extraction is required and the procedure can be carried out in Eppendorf tubes or 96-well microtiter plates. Starting with a phage supernatant, the entire procedure is carried out in the same reaction vessel and all separation steps are based on a novel application of magnetic bead separation. The design of a 96-well magnetic separator is presented and the application of the method for large-scale sequencing is discussed.     

A microtiter plate assay for N-acetyl-beta-D-glucosaminidase using a fluorogenic substrate

We describe a simple endpoint method for the determination of N-acetyl-beta-D-glucosaminidase (NAGase; EC 3.2.1.30). NAGase uses a fluorogenic substrate, 4-methylumbelliferyl-N-acetyl-beta-D-glucosaminide, at pH 4.6, liberating the fluorescent 4-methylumbelliferone. The method is reproducible and fast both at room temperature and at 37 degrees C. The procedure developed can be used, e.g., in the diagnosis of bovine subclinical mastitis, where elevated NAGase activities are found in raw milk samples. The assay procedure has a high capacity and high sensitivity and several hundred milk samples can be screened per hour using 96-well microtiter plates and an automated fluorescence reader. In addition to its use in mastitis diagnosis, the assay can be used in the diagnosis of some diseases of human origin.     

An automated method for DNA preparation from thousands of YAC clones.

We describe an automated method for the preparation of yeast genomic DNA capable of preparing thousands of DNAs in parallel from a YAC library. Briefly, the protocol involves four steps: (1) Yeast clones are grown in the wells of 96-well microtiter plates with filter (rather than plastic) well-bottoms, which are embedded in solid growth media; (2) These yeast cultures are resuspended and their concentrations determined by optical density measurement; (3) Equal numbers of cells from each well are embedded in low-melting temperature agarose blocks in fresh 96-well plates, again with filter bottoms; and (4) DNA is prepared in the agarose blocks by a protocol similar to that used for preparing DNA for pulsed-field gels, with the reagents being dialyzed through the (filter) bottoms of the microtiter plate. The DNA produced by this method is suitable for pulsed-field gel electrophoresis, for restriction enzyme digestion, and for the polymerase chain reaction (PCR). Using this protocol, we produced 3000 YAC strain DNAs in three weeks. This automated procedure should be extremely useful in many genomic mapping projects.     

Development and evaluation of an automated workstation for single nucleotide polymorphism discrimination using bacterial magnetic particles

We designed an automated workstation for magnetic particle-based single nucleotide polymorphism (SNP) discrimination of ALDH genotypes. Bacterial magnetic particles (BMPs) extracted from Magnetospirillum magneticum AMB-1 were used as DNA carriers. The principle for SNP discrimination in this study was based on fluorescence resonance energy transfer (FRET) between FITC (donor) and POPO-3 (acceptor) bound to double-stranded DNA. The workstation is equipped with a 96-way automated pipetter which collects and dispenses fluids as it moves in x- and z-directions. The platform contains a disposable tip rack station, a reagent vessel serving as a stock for POPO-3 and FITC-labeled probes and a reaction station for a 96-well microtiter plate. BMPs were collected by attaching a neodymium iron boron sintered (Nd-Fe-B) magnet on the bottom of the microtiter plate. This system permits the simultaneous heating and magnetic separation of 96 samples per assay. The genotypes ALDH2*1 and ALDH2*2 were discriminated by calculating the relative fluorescence intensities on BMPs.     

Automated kinetic assay of beta-galactosidase activity.

An automated kinetic assay for beta-galactosidase activity in Escherichia coli was developed to permit the measurement of many independent samples simultaneously. Bacteria are grown, lysed from without (by adsorption of a high multiplicity of bacteriophage T4) and assayed in microtiter plates with 96 wells. Absorbance data are collected and analyzed by computer. The growth and lysis procedure, apparatus and software used in this assay can be used for other spectrophotometric enzyme assays.     

From genes to proteins: high-throughput expression and purification of the human proteome

The development of high-throughput methods for gene discovery has paved the way for the design of new strategies for genome-scale protein analysis. Lawrence Livermore National Laboratory and Onyx Pharmaceuticals, Inc., have produced an automatable system for the expression and purification of large numbers of proteins encoded by cDNA clones from the IMAGE (Integrated Molecular Analysis of Genomes and Their Expression) collection. This high-throughput protein expression system has been developed for the analysis of the human proteome, the protein equivalent of the human genome, comprising the translated products of all expressed genes. Functional and structural analysis of novel genes identified by EST (Expressed Sequence Tag) sequencing and the Human Genome Project will be greatly advanced by the application of this high-throughput expression system for protein production. A prototype was designed to demonstrate the feasibility of our approach. Using a PCR-based strategy, 72 unique IMAGE cDNA clones have been used to create an array of recombinant baculoviruses in a 96-well microtiter plate format. Forty-two percent of these cDNAs successfully produced soluble, recombinant protein. All of the steps in this process, from PCR to protein production, were performed in 96-well microtiter plates, and are thus amenable to automation. Each recombinant protein was engineered to incorporate an epitope tag at the amino terminal end to allow for immunoaffinity purification. Proteins expressed from this system are currently being analyzed for functional and biochemical properties.     

Use of a microplate reader in an assay of glutathione reductase using 5,5'-dithiobis(2-nitrobenzoic acid)

The use of 96-well microtiter plates and a programmable microplate reader to measure glutathione reductase in an assay based on reduction of 5,5'-dithiobis(2-nitrobenzoic acid) by GSH generated from an excess of GSSG is described. Samples are prepared in 96-well plates and absorbance at 415 nm with a reference wavelength of 595 is determined every 30 s for 3 min. The rate of increase in absorbance is directly proportional to the amount of glutathione reductase in the sample. Activity in an unknown sample is determined from a standard curve. The assay is rapid and allows many small samples to be analyzed in replicates of two or more at the same time.     

A Microtiter Plate Assay for Screening Antioxidant Activity in Extracts of Marine Organisms

A novel microtiter plate assay was developed to determine the total peroxyl radical-trapping activity of antioxidants extracted from marine organisms by measuring the inhibition rate of dye-substrate oxidation. We compared use of dihydrorhodamine-123, dihydrofluorescein, and dichlorodihydrofluorescein as reduced substrates for oxidation by peroxyl radicals generated from 2,2-azobis(2-amidinopropane) dihydrochloride. The oxidation products of these highly reactive substrates are intensely colored dyes that absorb maximally in the wavelength region, _max = 489 to 512 nm, and their concentrations were determined photometrically using a 96-well, microtiter plate reader. The microtiter plate method provides for concurrent multisample analysis with automated data storage, regression analyses, and calculation of oxidation inhibition rates. Dihydrorhodamine was selected as the preferred substrate for screening crude extracts, and typical assay results are presented. Novel lead antioxidants are selected from active extracts by chromatographic analysis with electrochemical detection.     

Measurement of hydrocarbon-degrading microbial populations by a 96-well plate most-probable-number procedure

A 96-well microtiter plate most-probable-number (MPN) procedure was developed to enumerate hydrocarbondegrading microorganisms. The performance of this method, which uses number 2 fuel oil (F2) as the selective growth substrate and reduction of iodonitrotetrazolium violet (INT) to detect positive wells, was evaluated by comparison with an established 24-well microtiter plate MPN procedure (the Sheen Screen), which uses weathered North Slope crude oil as the selective substrate and detects positive wells by emulsification or dispersion of the oil. Both procedures gave similar estimates of the hydrocarbon-degrader population densities in several oil-degrading enrichment cultures and sand samples from a variety of coastal sites. Although several oils were effective substrates for the 96-well procedure, the combination of F2 with INT was best, because the color change associated with INT reduction was more easily detected in the small wells than was disruption of the crude oil slick. The method's accuracy was evaluated by comparing hydrocarbon-degrader MPNs with heterotrophic plate counts for several pure and mixed cultures. For some organisms, it seems likely that a single cell cannot initiate sufficient growth to produce a positive result. Thus, this and other hydrocarbon-degrader MPN procedures might underestimate the hydrocarbon-degrading population, even for culturable organisms.     

An automated filtration assay for protein kinase C ligands

[3H]Phorbol dibutyrate ([3H]PDBu) binding to soluble mouse brain protein kinase C (PKC) was established in a 96-well microtiter plate assay. [3H]PDBu-PKC receptor complexes were rapidly aspirated from wells, filtered, and washed onto glass fiber filter mats using an automated cell harvester. Results were compared to a modification of a previously described assay in which components were incubated in tubes, and manually delivered and washed onto filters with a manifold filtration apparatus. Both 96-well plate and tube assays gave qualitatively and quantitatively similar results since: (i) [3H]PDBu binding to PKC was phosphatidylserine (PS) dependent and calcium stimulatable; (ii) the amounts of [3H]PDBu bound by filters with each technique at receptors excess were similar, 3.2 +/- 0.3 and 3.1 +/- 0.4 pmol respectively; and (iii) the affinities of [3H]PDBu for PKC were comparable; Kd's were 1.95 +/- 0.3 and 2.2 +/- 0.55 nM, respectively. The 96-well plate assay was more accurate and rapid than the tube assay. The microtiter plate assay was adapted for use with [N,N-dimethyl-3H]N,N-dimethylstaurosporine ([3H]DMS). With [3H]PDBu and [3H]DMS as ligands, the 96-well plate method was used for the rapid discrimination of agents which bound selectively at the regulatory and/or catalytic domains of PKC.     

High-throughput screening with HyperCyt flow cytometry to detect small molecule formylpeptide receptor ligands

High-throughput flow cytometry (HTFC), enabled by faster automated sample processing, represents a promising high- content approach for compound library screening. HyperCyt is a recently developed automated HTFC analysis system by which cell samples are rapidly aspirated from microplate wells and delivered to the flow cytometer. The formylpeptide receptor (FPR) family of G protein-coupled receptors contributes to the localization and activation of tissue-damaging leukocytes at sites of chronic inflammation. Here, the authors describe development and application of an HTFC screening approach to detect potential anti-inflammatory compounds that block ligand binding to FPR. Using a homogeneous no-wash assay, samples were routinely processed at 1.5 s/well (approximately 2500 cells analyzed/sample), allowing a 96-well plate to be processed in less than 2.5 min. Assay sensitivity and accuracy were validated by detection of a previously documented active compound with relatively low FPR affinity (sulfinpyrazone, inhibition constant [K(i)]=14 microM) from among a collection of 880 compounds in the Prestwick Chemical Library. The HyperCyt system was therefore demonstrated to be a robust, sensitive, and highly quantitative method with which to screen lead compound libraries in a 96-well format.     

A simple, continuous fluorometric assay for HIV protease

Novel fluorogenic substrates for human immunodeficiency viral protease have been developed based on the principle of fluorescence energy transfer. Starting from a p24/p15 cleavage site-derived hexapeptide substrate. Ac-Thr-Ile-Nle-Nle-Gln-Arg-NH2, incorporation of 2-aminobenzoic acid in place of the acetyl group as the donor and p-NO2-Phe at the P1' position as acceptor gave the intramolecularly quenched fluorogenic substrate. Cleavage of the substrate by HIV protease released the fluorescent N-terminal tripeptide from its close apposition to the quenching nitrobenzyl group, resulting in enhanced fluorescence. An automated assay based on 96-well microtiter plates and a fluorometric plate reader have been developed, which allow high throughput of compounds in the search for HIV protease inhibitors.     

Potency assays for Anistreplase: comparison of the fibrin plate assay and a 96-well plate assay.

Several production lots of Anistreplase (Eminase) were assayed for potency by either two fibrin plate assays or a clot lysis assay performed in 96-well microtiter plates. The 96-well plate assay yielded comparable data to the fibrin plate assays and had the advantage of greater efficiency with respect to both time and reagents. As a result the newer method appears to be a suitable alternative to the fibrin plate assays for lot release of Anistreplase.     

Simple and rapid preparation of plasmid template by a filtration method using microtiter filter plates.

We developed a new simple high-throughput plasmid DNA extraction procedure, based on a modified alkaline lysis method, using only one 96-well microtiter glassfilter plate. In this method, cell harvesting, lysis by alkaline and plasmid purification are performed on only one microtiter glassfilter plate. After washing out RNAs or other contaminants, plasmid DNA is eluted by low-ion strength solution, although precipitated chromosomal DNA is not eluted. The plasmid prepared by this method can be applied to sequencing reactions or restriction enzyme cleavage.     

Comparison of plasma sample purification by manual liquid–liquid extraction, automated 96-well liquid–liquid extraction and automated 96-well solid-phase extraction for analysis by high-performance liquid chromatography with tandem mass spectrometry

Three extraction procedures were developed for the quantitative determination of a carboxylic acid containing analyte (I) in human plasma by high-performance liquid chromatography (HPLC) with negative ion electrospray tandem mass spectrometry (MS–MS). The first procedure was based on the manual liquid–liquid extraction (LLE) of the acidified plasma samples with methyl tert.-butyl ether. The second procedure was based on the automation of the manual LLE procedure using 96-well collection plates and a robotic liquid handling system. The third approach was based on automated solid-phase extraction (SPE) using 96-well SPE plates and a robotic liquid handling system. A lower limit of quantitation of 50 pg/ml was achieved using all three extraction procedures. The total time required to prepare calibration curve standards, aliquot the standards and plasma samples, and process a total of 96 standards and samples by manual LLE was three-times longer than the time required for 96-well SPE or 96-well LLE (4 h, 50 min vs. 1 h, 43 min). Even more importantly, the time the bioanalyst physically spent on the 96-well LLE or 96-well SPE procedure was only a small fraction of the time spent on the manual LLE procedure (<10 min vs. 4 h, 10 min). It should be noted that the 96-well SPE procedure incorporated the two steps of evaporation of the eluates to dryness and subsequent reconstitution of the dried extract. The total time required for the 96-well SPE could be reduced by 50% if the eluates were injected directly, eliminating the drying and reconstitution steps, which is achievable when sensitivity is less of an issue.     

A multi-well filtration assay for quantitation of inositol phosphates in biological samples

The quantitation of inositol phosphates (IPs), mediators of certain signal transduction processes, typically involves laborious and time consuming conventional ion-exchange chromatography procedures. We have developed a high throughput microtiter plate-based IP assay that utilizes vacuum rather than gravitational flow and has significant advantages over existing methods. The response of recombinant HEK-293 cells expressing human LHRH receptor cDNA to LHRH agonists was used as a model system to develop the assay conditions. Cell lysates containing labeled IPs were applied in 96-well plates fitted with filtration discs containing regenerated Dowex AGI-X8 resin. Specifically bound inositol phosphates were eluted with 1 M ammonium formate in 0.1 M formic acid directly into a fresh 96-well plate and an aliquot of the eluate from each well is transferred into a 96-well plate and counted. The results were comparable to those obtained with the conventional column method and the variation among replicates was significantly improved. This assay facilitates rapid quantitation of inositol phosphates from a large number of samples with relative ease and reduced generation of radioactive waste.