96-Well plate assays for measuring collagenase activity using (3)H-acetylated collagen.

We describe two alternative assays for measuring collagenolytic activity using (3)H-acetylated collagen. Both assays have been developed for the 96-well plate format and measure the amount of radiolabeled collagen fragments released into the supernatant from an insoluble (3)H-acetylated collagen fibril preparation. The first method separates digested solubilized fragments from the intact fibril by sedimentation of the undigested collagen by centrifugation. The second method achieves this separation by filtration of the supernatant through the membrane of a 96-well filtration plate which retains the undigested collagen fibril. Both methods give linear dose- and time-dependent responses of collagenase activity > or = 70% of total collagen lysis. In addition, both assays can be simply modified to measure tissue inhibitors of metalloproteinases (TIMPs) inhibitory activity, which is also linear between 20 and 75% of total collagen lysis with the amount of TIMP added.     

Investigation of high-throughput ultrafiltration for the determination of an unbound compound in human plasma using liquid chromatography and tandem mass spectrometry with electrospray ionization

A high-throughput ultrafiltration method with a direct injection assay has been developed to determine unbound concentrations of a high-protein binding compound, an alpha(v)beta(3) bone integrin antagonist (I), in human plasma for a clinical pharmacokinetic study. The 96-well MultiScreen filter plate with Ultracel-PPB membrane was evaluated for the separation of unbound from protein-bound compound I by ultrafiltration. The sample preparation was automated using a Packard MultiPROBE II EX liquid handling system to transfer the plasma samples to the 96-well PPB plate for centrifugation and to prepare ultrafiltrate samples for analysis. Using on-line extraction with a column-switching setup for sample clean-up and separation, the ultrafiltrate samples were directly injected onto a reversed-phase HPLC system and analyzed using a mass spectrometer interfaced with an electrospray ionization (ESI) source in the positive ionization mode (LC/ESI-MS/MS). The performance of the ultrafiltration using Ultracel-PPB 96-well plate for unbound I analysis was evaluated and optimized with respect to sample volume, centrifugation temperature, speed and time, and the relationship of the well positions of the PPB plate versus filtrate volumes and concentrations. The assay intraday accuracy and precision were between 93.9 and 104.8 and <7.3% (CV), respectively. The linear range of the calibration curve for the assay was 0.1-500 ng/mL on a Finnigan TSQ Quantum LC/ESI-MS/MS system. Evaluation and validation of the unbound plasma assay demonstrated it to be rapid, sensitive and reproducible.     

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.     

Coated microwell plate-based affinity purification of antigens

Antibody-based affinity capture of antigens is widely used in the isolation of antigens from complex mixtures. Antibody and the corresponding antigen are allowed to interact with each other to form immunocomplexes which are then typically captured by protein A or protein G immobilized on beaded support. Antigen capture performed using this method generally requires multiple centrifugation steps and careful pipetting to avoid loss of the bead-bound complexes. This traditional procedure is tedious and not easily reproducible, especially when working with multiple samples. To address these issues we have demonstrated that antigens can be captured with protein A/G, protein G, and high binding-capacity streptavidin 96-well strip-coated plates. The coated plate method of antigen purification is reproducible, within the same experiment and between experiments, due to the uniform binding capacity of the plates and wells. Here we report the use of coated microwell plates for antigen capture and for protein-protein interaction studies with the well-characterized BIR2-SMAC, transferrin receptor/ transferrin, and other systems.     

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.     

Use of trifluoroacetic acid to quantify small, polar compounds in rat plasma during discovery-phase pharmacokinetic evaluation

Although it is accepted that trifluoroacetic acid (TFA) can cause suppression of an analyte during LC/MS analysis, this paper presents a relatively sensitive gradient method that uses a TFA mobile phase for the improved quantification of small, polar drug-like compounds. The described method was developed in a discovery drug metabolism and pharmacokinetics (DMPK) laboratory for the screening measurement of compound concentrations to calculate PK parameters and CNS exposure of compounds from a chemical series that had poor chromatography under generic methods using formic acid mobile phase. The samples were collected by a Culex automated sampling unit, and the plasma proteins were precipitated by a Tecan robot in 96-well plates. After centrifugation, the supernatant was removed, dried down using a SPE-Dry unit, and the samples were reconstituted in aqueous buffer on the robot. The samples were analyzed on an Agilent LC/MSD using a 5-min gradient on a 5 cm phenyl column. No additional steps, such as the "TFA-fix", were necessary. Although sample batches were analyzed over 6h, no drift or degradation of signal was observed. The improved chromatography resulted in a method that was selective, rugged, and had a dynamic range from 5 to 20,000 nM, which was sufficient to quantitate low volume, serial plasma samples collected out to 8 h postdose.     

Simplified method for determination of clarithromycin in human plasma using protein precipitation in a 96-well format and liquid chromatography-tandem mass spectrometry

A simplified method to determine clarithromycin concentrations in human plasma using protein precipitation in a 96-well plate and liquid chromatography-tandem mass spectrometry was developed and validated. Plasma proteins were precipitated with acetonitrile and roxithromycin was used as the internal standard. After vortex mixing and centrifugation, the supernatants were directly injected onto a Phenomenex Luna Phenyl-Hexyl column (50 mm x 2.0 mm ID, 3 microm). The mobile phase consisted of water and methanol (30:70, v/v) containing 0.1% formic acid and 5mM ammonium acetate. The flow rate was 0.22 mL/min and the total run time (injection to injection) was less than 3 min. Detection of the analytes was achieved using positive ion electrospray tandem mass spectrometry in selected reaction monitoring (SRM) mode. The linear standard curve ranged from 100 to 5000 ng/mL and the precision and accuracy (inter- and intra-run) were within 7.9% and 4.9%, respectively. The method was successfully used to determine clarithromycin concentrations in human plasma samples obtained from healthy subjects who were given clarithromycin 500 mg for 3 days. The method is rapid, simple, precise and directly applicable to clarithromycin pharmacokinetic studies.     

Comparison of two methods for rapid detection of cytomegalovirus viraemia in cell culture

Two methods for rapid CMV viraemia detection in cell culture with immunoperoxidase staining performed 40 h post-inoculation were compared, on 230 blood samples: (1) a 24-well plate centrifugation assay and (2) a 25-cm²-culture flask technique, without centrifugation. The flask technique was significantly more sensitive than the 24-well plate technique. Moreover, the flask assay was more cost-effective and offered other advantages such as easier sterile handling and less damage of the cell monolayer.     

Evaluation of online extraction/mass spectrometry for in vivo cassette analysis

An online extraction/mass spectrometry technique was evaluated for direct analysis of plasma samples. A simple user-friendly online extraction system that consists of two pumps, an autosampler, a six-port switching valve and a mass spectrometer is described. The system was controlled by the LC-MS software (Masslynx 3.5, Waters Corporation, Beverly, MA). Various analytical conditions such as extraction column, mobile phases, run time and wash solvent were optimized to establish an analytical method that was simple, easy to set up and generic. Sample preparation effort was minimal, which included dilution of plasma with water and centrifugation conducted in 96-well plate format. The system was used to analyze in vivo plasma samples from rat n-in-one cassette dosing studies. Concentration and pharmacokinetic (PK) data obtained from the online extraction method were comparable with data obtained from the protein precipitation extraction method. Overall, the simple, robust online extraction system provides cost savings by minimizing sample preparation and method development time. The system was used to analyze compounds from different structural classes. These studies suggest that calculated lipophilicity of a compound can be used as a tool for pre-selection of extraction column, which would save method development time for early discovery studies.     

Targeting the MraY and MurG bacterial enzymes for antimicrobial therapeutic intervention

Assays for two enzymes from Escherichia coli were developed and validated as antibacterial inhibitor screens. The MraY and MurG enzymes were overexpressed and purified as the membrane fraction or to homogeneity, respectively. The MurG enzyme was expressed with a six-histidine tag using an optimized minimal-medium protocol for subsequent purification. Although traditional assays were established, the enzymes were also assayed via a 96-well membrane plate assay and a 384-well scintillation proximity-based assay developed herein. These assays afford a more economical and high-throughput evaluation of inhibitors. A mureidomycin inhibitor mix was used as a control for the assay development and screen validation. Several inhibitors resulting from a high-throughput screen were found and evaluated for potential therapeutic use.     

Protocols and Programs for High-Throughput Growth and Aging Phenotyping in Yeast

In microorganisms, and more particularly in yeasts, a standard phenotyping approach consists in the analysis of fitness by growth rate determination in different conditions. One growth assay that combines high throughput with high resolution involves the generation of growth curves from 96-well plate microcultivations in thermostated and shaking plate readers. To push the throughput of this method to the next level, we have adapted it in this study to the use of 384-well plates. The values of the extracted growth parameters (lag time, doubling time and yield of biomass) correlated well between experiments carried out in 384-well plates as compared to 96-well plates or batch cultures, validating the higher-throughput approach for phenotypic screens. The method is not restricted to the use of the budding yeast Saccharomyces cerevisiae, as shown by consistent results for other species selected from the Hemiascomycete class. Furthermore, we used the 384-well plate microcultivations to develop and validate a higher-throughput assay for yeast Chronological Life Span (CLS), a parameter that is still commonly determined by a cumbersome method based on counting “Colony Forming Units”. To accelerate analysis of the large datasets generated by the described growth and aging assays, we developed the freely available software tools GATHODE and CATHODE. These tools allow for semi-automatic determination of growth parameters and CLS behavior from typical plate reader output files. The described protocols and programs will increase the time- and cost-efficiency of a number of yeast-based systems genetics experiments as well as various types of screens.     

A derivative of diethylenetriaminepentaacetic acid for europium labeling of proteins.

Preparation of a reagent that will incorporate diethylenetriaminepentaacetic acid (DTPA) into proteins under mild conditions and make a strong europium chelate is described. Aminoacetaldehyde diethyl acetal was reacted with DTPA dianhydride, and mono- and disubstituted products as well as unsubstituted DTPA were separated by gel filtration. The monosubstituted product, after conversion into the corresponding aldehyde by mild acid hydrolysis, is conjugated to protein or other amino-containing compounds via reductive amination at neutral pH. Although the DTPA-Eu-labeled proteins are themselves not fluorescent, a strong fluorescence of europium can be generated easily by the dissociation-enhancement mechanism. A direct measurement of lectin-ligand interaction using Eu-labeled ligand and lectin immobilized on 96-well plate illustrates that the assay utilizing Eu fluorescence is as sensitive as the radioactive assays.     

Development of a high throughput 96-well plate sample preparation method for the determination of trileptal (oxcarbazepine) and its metabolites in human plasma

A high throughput preparation method for the determination of trileptal (oxcarbazepine, OXC) and its mono (MHD) and dihydroxy (DHD) metabolites in human plasma, using 96-well plate technology, has been developed and validated according to international regulatory requirements. Preparation of plasma samples (50 μl) containing the compounds to be analysed involved solid-phase extraction (SPE) on Empore C₁₈ 96-well SPE plates. Eluates from the plate were injected onto a reversed-phase column (Hypersil C₁₈, 3 μm) with UV detection at 210 nm. Detector response was linear over the ranges 0.2–10, 0.1–200 and 0.1–20 μmol/l, for OXC, MHD and DHD, respectively, with relative standard deviations from 1 to 10% and mean accuracies within 4% of the nominal values (number of standard curves=3 in duplicate). The limits of quantitation were 0.2, 0.1 and 0.1 μmol/l, respectively. The overall mean accuracies ranged from 96 to 106% and precision was in the range 4 to 11%. Cross validation indicated no significant difference between plasma concentrations obtained using the 96-well method and the previous method using a traditional SPE method with a 50 mg C₁₈ cartridge. About a threefold increase in sample throughput and a twofold decrease of plasma volume required for the assays, were the main advantages obtained from the previous method. The method was applied for the determination of 3000 plasma samples from clinical studies.     

An efficient, automatable template preparation for high throughput sequencing

We have developed a 96-well format for DNA template isolation that can be readily automatable. The template isolation protocol involves simple alkaline lysis chemistry and reversible capture on a silica solid phase. After the cells are lysed, no centrifugation is necessary, as lysate purification, DNA binding, washing, and release occur in 96-well filter plates. Large numbers of templates prepared using the silica purification method have been sequenced and analyzed. The quality of sequence resulting from our method has been compared with that generated from several commercial plasmid preparation protocols. We found sequence quality of the silica bead preparations to be equivalent to or, in some cases, better than those prepared by other methods. This method offers many advantages over other protocols we have used. First, the silica purifications have allowed us to more than double overall laboratory throughput while decreasing our template isolation materials cost at least five-fold. Second, because we have eliminated all centrifugation steps in the protocol, automation has been much simpler. The protocol has also been adapted to purify PCR products for use as templates in subsequent sequencing reactions.     

Automated liquid-liquid extraction based on 96-well plate format in conjunction with ultra-performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) for the quantitation of methoxsalen in human plasma

A sensitive, specific and high throughput bioanalytical method using automated sample processing via 96-well plate liquid-liquid extraction and ultra-performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) has been developed for the determination of methoxsalen in human plasma. Plasma samples with ketoconazole as internal standard (IS) were prepared by employing 0.2mL human plasma in ethyl acetate:dichloromethane (80:20, v/v). The chromatographic separation was achieved on a Waters Acquity UPLC BEH C18 column using isocratic mobile phase, consisting of 10mM ammonium formate and acetonitrile (60:40, v/v), at a flow rate of 0.5mL/min. The linear dynamic range was established over the concentration range 1.1-213.1ng/mL for methoxsalen. The method was rugged and rapid with a total run time of 1.5min. It was successfully applied to a pivotal bioequivalence study in 12 healthy human subjects after oral administration of 10mg extended release methoxsalen formulation under fasting condition.     

Semi-automated 96-well solid-phase extraction and gas chromatography–negative chemical ionization tandem mass spectrometry for the trace analysis of fluprostenol in rat plasma

Semi-automated 96-well plate solid-phase extraction (SPE) was used for sample preparation of fluprostenol, a prostaglandin analog, in rat plasma prior to detection by gas chromatography–negative chemical ionization tandem mass spectrometry (GC–NCI-MS–MS). A liquid handling system was utilized for all aspects of sample handling prior to SPE including transferring of samples into a 96-well format, preparation of standards as well as addition of internal standard to standards, quality control samples and study samples. SPE was performed in a 96-well plate format using octadecylsilane packing and the effluent from the SPE was dried in a custom-made 96-well apparatus. The sample residue was derivatized sequentially with pentafluorobenzylbromide followed by N-methyl-N-trimethylsilyltrifluoroacetamide. The derivatized sample was then analyzed using GC–NCI-MS–MS. The dynamic range for the method was from 7 to 5800 pg/ml with a 0.1-ml plasma sample. The methodology was evaluated over a 4-day period and demonstrated an accuracy of 90–106% with a precision of 2.4–12.9%.     

Determination of morphine and morphine glucuronides in human plasma by 96-well plate solid-phase extraction and liquid chromatography-electrospray ionization mass spectrometry

There is considerable interest in quantifying morphine and its major metabolites, morphine-3-glucuronide (M3G) and morphine-6-glucuronide (M6G). Available assays use gas chromatography-mass spectrometry or high-performance liquid chromatography (HPLC) with single or tandem mass spectrometry, ultraviolet, electrochemical, or fluorimetric detection. Nevertheless, few methods provide adequate sensitivity for all analytes, in a single injection, with the desired rate of sample throughput. A rapid and sensitive method for quantification of morphine, M3G and M6G from human plasma using HPLC with electrospray ionization mass spectrometry was developed using a Waters Oasis MCX 96-well plate for extracting both lipophilic morphine and its hydrophilic glucuronides, C18 separation using an isocratic mobile phase (methanol, acetonitrile and formic acid), and selected ion monitoring. Recoveries of morphine, M3G and M6G, respectively, were 81, 90 and 82% at the low (2, 25 and 2 ng/ml), 80, 77 and 75% at the medium (10, 250 and 10 ng/ml), and 74, 62 and 72% at the high (100, 1000 and 100 ng/ml) quality control samples. The limit of quantitation was 0.5 ng/ml morphine and M6G, and 5 ng/ml M3G. Analytes were validated over a linear range of 0.5-200 ng/ml morphine and M6G, and 5-2000 ng/ml M3G. This assay represents an improvement over existing methods through solid phase extraction with increased sample throughput (96-well plates), use of small samples (0.5 ml), and sub-nanogram detection.     

Use of a 96-well microplate reader for measuring routine enzyme activities

A method is described for the routine determination of the rate of colorimetric enzyme reactions using a 96-well microtiter plate reader commonly used in immunoassay. This approach is illustrated by monitoring esterase activity using three common products: release of thiol, release of ethanol, and release of p-nitrophenylate ion. Examples include monitorings of the rate of hydrolysis of acetylthiocholine iodide by eel acetylcholinesterase and the rate of hydrolysis of malathion and nonconventional esters such as O-methyl, O-ethyl, and O-isobutyl carbonates of p-nitrophenol by commercial porcine liver carboxylesterase. Data obtained from the plate reader were compared to those obtained, under similar conditions, in a conventional spectrophotometer. Absorbance measurements made in both machines on the same solution, as well as absorbance changes measured over time, were similar. The use of the 96-well plate format tremendously increased the number of enzyme assays carried out per person and the interface with a personal computer allowed rapid manipulation of the absorbance values to calculate the desired rate data. This approach should be generally applicable to many routine colorimetric assays in the research laboratory.     

Ice-Cap: A Method for Growing Arabidopsis and Tomato Plants in 96-well Plates for High-Throughput Genotyping

It is becoming common for plant scientists to develop projects that require the genotyping of large numbers of plants. The first step in any genotyping project is to collect a tissue sample from each individual plant. The traditional approach to this task is to sample plants one-at-a-time. If one wishes to genotype hundreds or thousands of individuals, however, using this strategy results in a significant bottleneck in the genotyping pipeline. The Ice-Cap method that we describe here provides a high-throughput solution to this challenge by allowing one scientist to collect tissue from several thousand seedlings in a single day ^1,2. This level of throughput is made possible by the fact that tissue is harvested from plants 96-at-a-time, rather than one-at-a-time.The Ice-Cap method provides an integrated platform for performing seedling growth, tissue harvest, and DNA extraction. The basis for Ice-Cap is the growth of seedlings in a stacked pair of 96-well plates. The wells of the upper plate contain plugs of agar growth media on which individual seedlings germinate. The roots grow down through the agar media, exit the upper plate through a hole, and pass into a lower plate containing water. To harvest tissue for DNA extraction, the water in the lower plate containing root tissue is rapidly frozen while the seedlings in the upper plate remain at room temperature. The upper plate is then peeled away from the lower plate, yielding one plate with 96 root tissue samples frozen in ice and one plate with 96 viable seedlings. The technique is named "Ice-Cap" because it uses ice to capture the root tissue. The 96-well plate containing the seedlings can then wrapped in foil and transferred to low temperature. This process suspends further growth of the seedlings, but does not affect their viability. Once genotype analysis has been completed, seedlings with the desired genotype can be transferred from the 96-well plate to soil for further propagation. We have demonstrated the utility of the Ice-Cap method using Arabidopsis thaliana, tomato, and rice seedlings. We expect that the method should also be applicable to other species of plants with seeds small enough to fit into the wells of 96-well plates.     

Adaptation of aequorin functional assay to high throughput screening

AequoScreen, a cellular aequorin-based functional assay, has been optimized for luminescent high-throughput screening (HTS) of G protein-coupled receptor (GPCRs). AequoScreen is a homogeneous assay in which the cells are loaded with the apoaequorin cofactor coelenterazine, diluted in assay buffer, and injected into plates containing the samples to be tested. A flash of light is emitted following the calcium increase resulting from the activation of the GPCR by the sample. Here we have validated a new plate reader, the Hamamatsu Photonics FDSS6000, for HTS in 96- and 384-well plates with CHO-K1 cells stably coexpressing mitochondrial apoaequorin and different GPCRs (AequoScreen cell lines). The acquisition time, plate type, and cell number per well have been optimized to obtain concentration-response curves with 4000 cells/well in 384-well plates and a high signal:background ratio. The FDSS6000 and AequoScreen cell lines allow reading of twenty 96- or 384-well plates in 1 h with Z' values of 0.71 and 0.78, respectively. These results bring new insights to functional assays, and therefore reinforce the interest in aequorin-based assays in a HTS environment.