A homogeneous and multiplexed immunoassay for high-throughput screening using fluorometric microvolume assay technology.

We have developed a simple, homogeneous bead-based immunoassay for use with fluorometric microvolume assay technology (FMAT). The FLISA (fluorescence-linked immunosorbent assay) can be easily adapted from existing immunoassays, is comparable to traditional ELISAs with respect to linear dynamic range and sensitivity, and can be readily performed in 96- and 384-well plates. Additionally, the FLISA utilizes 100-fold less primary antibody than the conventional immunoassay. The scanner uses a helium/neon laser to image and measure bead-bound fluorescence while the background fluorescence is ignored. Consequently, no wash steps are required to remove unbound antibody, ligand, and fluorophore. Furthermore, the instrument is capable of detecting two different fluorescent dyes, allowing for multiplexed assays based on color. Fluorescent bead-based immunoassays were developed for the cytokines IL-6 and IL-8, and their use in both one-color and two-color FLISAs is demonstrated. Although no wash steps were employed, the FLISA was able to accurately measure the concentrations of IL-6 and IL-8 in the growth media of cytokine-stimulated HUVEC cells. In addition, a simulated high-throughput two-color FLISA positively identified those wells in a 384-well plate that contained different amounts of IL-6 and/or IL-8 peptide. The homogeneous, multiplex and multiplate format of the FLISA reduces hands-on time and reagent usage, and is therefore ideally suited for high-throughput screening.     

A high-throughput hybridoma selection method using fluorometric microvolume assay technology

Monoclonal antibodies (mAb) are not only useful reagents but also represent a promising type of therapeutics due to their high affinity and exquisite specificity for their antigens. A critical step in mAb generation is to identify antigen-specific antibodies. Although enzyme-linked immunosorbent assay (ELISA) has been broadly applied for antibody selection against secreted antigens, an inherent disadvantage for ELISA is the difficulty in identifying antibodies that recognize the native conformation of cell surface antigens. To overcome this drawback, the authors have developed a high-throughput cell-based antibody binding assay using fluorometric microvolume assay technology (FMAT). This method offers a homogeneous assay for detection of antibody binding to its antigen on the cell surface. To distinguish antibodies that bind to antigen on the cell surface from those that bind nonspecifically to cells, the binding is assessed using both antigen-expressing cells and related cells devoid of the antigen expression. This assay can detect antibodies at a concentration as low as 5 ng/mL and cell surface antigen as low as 9000 copies per cell. Results demonstrate that the FMAT method provides a sensitive and homogeneous assay to detect antibody binding to cell surface antigens and is amenable for high-throughput hybridoma selection.     

A homogeneous,nonradioactive high-throughput fluorogenic protein kinase assay

Protein kinases play an important role in many cellular processes and mediate cellular responses to a variety of extracellular stimuli. They have been identified by many pharmaceuticals as valid targets for drug discovery. Because of the large number of protein kinases, and the large number of compounds to be screened, it is important to develop assay systems that are not only sensitive but also homogeneous, fast, simple, nonradioactive, and cost-effective. Here we present a novel, rapid, robust assay to measure the enzyme activity of low concentrations of several serine/threonine and tyrosine protein kinases. It is based on the use of fluorogenic peptide substrates (Rhodamine 110, bis peptide amide) that are cleaved before phosphorylation to release the free Rhodamine 110; upon phosphorylation, cleavage is hindered, and the compound remains as a nonfluorescent peptide conjugate. The assay can be carried out in single- as well as multiwell plate formats such as 96- and 384-well plates. The signal-to-noise ratio is very high (40), the Z(') is over 0.8, and the signal is stable for at least 4h. Finally, the assay is easily adapted to a robotic system for drug discovery programs targeting protein kinases.     

A homogeneous, nonradioactive high-throughput fluorogenic protein phosphatase assay

Protein phosphatases are critical components in cellular regulation; they do not only act as antioncogenes by antagonizing protein kinases, but they also play a positive regulatory role in a variety of cellular processes that require dephosphorylation. Thus, assessing the function of these enzymes necessitates the need for a robust, sensitive assay that accurately measures their activities. The authors present a novel, homogeneous, and nonradioactive assay to measure the enzyme activity of low concentrations of several protein phosphatases (phosphoserine/phosphothreonine phosphatases and phosphotyrosine phosphatases). The assay is based on the use of fluorogenic peptide substrates (rhodamine 110, bis-phosphopeptide amide) that do not fluoresce in their conjugated form, which is resistant to cleavage by aminopeptidases. However, upon dephosphorylation by the phosphatase of interest, the peptides become cleavable by the protease and release the highly fluorescent-free rhodamine 110. The assay is rapid, can be completed in less than 2 h, and can be carried out in multiwell plate formats such as 96-, 384-, and 1536-well plates. The assay has an excellent dynamic range, high signal-to-noise ratio, and a Z' of more than 0.8, and it is easily adapted to a robotic system for drug discovery programs targeting protein phosphatases.     

A nonradioactive DNA methyltransferase assay adaptable to high-throughput screening

We have developed a nonradioactive assay method for DNA methyltransferases based on the ability to protect substrate DNA from restriction. DNA immobilized to a microplate well was treated sequentially with methyltransferase and an appropriate endonuclease. The amount of methylated DNA product is reflected by a proportional decrease in endonuclease cleavage, which is in turn reflected by increased retention of the end-labeled affinity probe. A single universal substrate was designed to assay multiple methyltransferases including those that do not have a cognate endonuclease. The methodology developed is suited to screen a large number of compounds for inhibitors of various methyltransferases.     

A novel multiplex cell viability assay for high-throughput RNAi screening

Cell-based high-throughput RNAi screening has become a powerful research tool in addressing a variety of biological questions. In RNAi screening, one of the most commonly applied assay system is measuring the fitness of cells that is usually quantified using fluorescence, luminescence and absorption-based readouts. These methods, typically implemented and scaled to large-scale screening format, however often only yield limited information on the cell fitness phenotype due to evaluation of a single and indirect physiological indicator. To address this problem, we have established a cell fitness multiplexing assay which combines a biochemical approach and two fluorescence-based assaying methods. We applied this assay in a large-scale RNAi screening experiment with siRNA pools targeting the human kinome in different modified HEK293 cell lines. Subsequent analysis of ranked fitness phenotypes assessed by the different assaying methods revealed average phenotype intersections of 50.7±2.3%-58.7±14.4% when two indicators were combined and 40-48% when a third indicator was taken into account. From these observations we conclude that combination of multiple fitness measures may decrease false-positive rates and increases confidence for hit selection. Our robust experimental and analytical method improves the classical approach in terms of time, data comprehensiveness and cost.     

A nonradioactive screening method for cloning genes encoding sequence-specific DNA binding proteins.

We have developed a novel nonradioactive screening method for cloning genes encoding sequence-specific DNA binding proteins. This method is derived from previously described protocols developed for the same purpose by using radioactively labeled DNA probes containing protein recognition sequences. This nonradioactive strategy relies upon the use of a small hapten, digoxigenin. Fusion proteins expressed from the recombinant bacteriophage lambda gt11/lambda ZAP are immobilized on nitrocellulose filters and probed with digoxigenin-labeled double-stranded DNA as a ligand. The specifically bound DNA probes can be detected through sequential incubations with antibody-enzyme conjugate and enzyme substrates. This technique has been successfully utilized to isolate several cDNA clones encoding DNA binding proteins.     

A nonradioactive high-throughput assay for screening and characterization of adenylation domains for nonribosomal peptide combinatorial biosynthesis

Adenylation domains are critical enzymes that dictate the identity of the amino acid building blocks to be incorporated during nonribosomal peptide (NRP) biosynthesis. NRPs display a wide range of biological activities and are some of the most important drugs currently used in clinics. Traditionally, activity of adenylation domains has been measured by radioactive ATP-[³²P]pyrophosphate (PP_i) exchange assays. To identify adenylation domains for future combinatorial production of novel NRPs as potential drugs, we report a convenient high-throughput nonradioactive method to measure activity of these enzymes. In our assay, malachite green is used to measure orthophosphate (P_i) concentrations after degradation by inorganic pyrophosphatase of the PP_i released during aminoacyl-AMP formation by action of the adenylation domains. The assay is quantitative, accurate, and robust, and it can be performed in 96- and 384-well plate formats. The performance of our assay was tested by using NcpB-A₄, one of the seven adenylation domains involved in nostocyclopeptide biosynthesis. The kinetics of pyrophosphate release monitored by this method are much slower than those measured by a traditional ATP-[³²P]PP_i exchange assay. This observation indicates that the formation of the adenylated amino acid and its release are the rate-limiting steps during the catalytic turnover.     

A high-throughput screening method for amino acid dehydrogenase

A simple and rapid screening method for amino acid dehydrogenase (e.g., leucine dehydrogenase, LDH) has been developed. It relies on a competitive relationship between a non-fluorescent Cu(II)–calcein complex and amino acid (e.g., l-2-aminobutyric acid, l-ABA). When ABA was introduced to a Cu(II)–calcein solution, it bound with the Cu(II) ions and this released calcein from the complex, which was detected as strong fluorescence. The principle of this high-throughput screening method was validated by screening an LDH mutant library. Compared with other methods, this method provided much quicker l-ABA detection and screening for leucine dehydrogenase mutations.     

A high-throughput screening for phosphatases using specific substrates

A high-throughput screening was developed for the detection of phosphatase activity in bacterial colonies. Unlike other methods, the current procedure can be applied to any phosphatase because it uses physiological substrates and detects the compelled product of all phosphatase reactions, that is, orthophosphate. In this method, substrates diffuse from a filter paper across a nitrocellulose membrane to bacterial colonies situated on the opposite face, and then reaction products flow back to the paper. Finally, a colorimetric reagent discloses the presence of orthophosphate in the filter paper. We validated the performance of this assay with several substrates and experimental conditions and with different phosphatases, including a library of randomly mutagenized rapeseed chloroplast fructose-1,6-bisphosphatase. This procedure could be extended to other enzymatic activities provided that an appropriate detection of reaction products is available.     

A generic high-throughput screening assay for kinases: protein kinase a as an example

A generic high-throughput screening assay based on the scintillation proximity assay technology has been developed for protein kinases. In this assay, the biotinylated (33)P-peptide product is captured onto polylysine Ysi bead via avidin. The scintillation signal measuring the product formation increases linearly with avidin concentration due to effective capture of the product on the bead surface via strong coulombic interactions. This novel assay has been optimized and validated in 384-well microplates. In a pilot screen, a signal-to-noise ratio of 5- to 9-fold and a Z' factor ranging from 0.6 to 0.8 were observed, demonstrating the suitability of this assay for high-throughput screening of random chemical libraries for kinase inhibitors.     

Homogeneous and nonradioactive high-throughput screening platform for the characterization of kinase inhibitors in cell lysates

Protein kinases are directly implicated in many human diseases; therefore, kinase inhibitors show great promises as new therapeutic drugs. In an effort to facilitate the screening and the characterization of kinase inhibitors, a novel application of the AlphaScreen technology was developed to monitor JNK activity from (1) purified kinase preparations and (2) endogenous kinase from cell lysates preactivated with different cytokines. The authors confirmed that both adenosine triphosphate (ATP) competitive as well as peptide-based JNK inhibitors were able to block the activity of both recombinant and HepG2 endogenous JNK activity. Using the same luminescence technique adapted for binding studies, the authors characterized peptide inhibitor mechanisms by measuring the binding affinity of the inhibitors for JNK. Because of the versatility of the technology, this cell-based JNK kinase assay could be adapted to other kinases and would represent a powerful tool to evaluate endogenous kinase activity and test a large number of potential inhibitors in a more physiologically relevant environment.     

A homogeneous enzyme fragment complementation-based beta-arrestin translocation assay for high-throughput screening of G-protein-coupled receptors

G-protein-coupled receptors (GPCRs) represent one of the largest gene families in the human genome and have long been regarded as valuable targets for small-molecule drugs. The authors describe a new functional assay that directly monitors GPCR activation. It is based on the interaction between beta-arrestin and ligand-activated GPCRs and uses enzyme fragment complementation technology. In this format, a GPCR of interest is fused to a small (approximately 4 kDa), optimized alpha fragment peptide (termed ProLink) derived from beta-galactosidase, and beta-arrestin is fused to an N-terminal deletion mutant of beta-galactosidase (termed the enzyme acceptor [EA]). Upon activation of the receptor, the beta-arrestin-EA fusion protein binds the activated GPCR. This interaction drives enzyme fragment complementation, resulting in an active beta-galactosidase enzyme, and thus GPCR activation can be determined by quantifying beta-galactosidase activity. In this report, the authors demonstrate the utility of this technology to monitor GPCR activation and validate the approach using a Galphai-coupled GPCR, somatostatin receptor 2. Potential application to high-throughput screens in both agonist and antagonist screening modes is exemplified.     

Screening for ligands using a generic and high-throughput light-scattering-based assay

Rapid identification of small molecules that interact with protein targets using a generic screening method greatly facilitates the development of therapeutic agents. The authors describe a novel method for performing homogeneous biophysical assays in a high-throughput format. The use of light scattering as a method to evaluate protein stability during thermal denaturation in a 384-well format yields a robust assay with a low frequency of false positives. This novel method leads to the identification of interacting small molecules without the addition of extraneous fluorescent probes. The analysis and interpretation of data is rapid, with sensitivity for protein stability comparable to differential scanning calorimetry. The authors propose potential uses in drug discovery, structural genomics, and functional genomics as a method to evaluate small-molecule interactions, identify natural cofactors that stabilize target proteins, and identify natural substrates and products for previously uncharacterized protein targets.     

A homogeneous high throughput nonradioactive method for measurement of functional activity of Gs-coupled receptors in membranes

A method is described for measuring the activity of G(s)-coupled receptors in a nonradioactive homogeneous membrane-based assay. This method has several major advantages over currently used methods for measuring functional activity of G(s)-coupled receptors. The assay is high throughput (>150,000 data points/day using a single reader). Dimethyl sulfoxide tolerance is high ( approximately 10%). Compared to complex cell-based assays, there is limited potential for nonspecific compound action. This resulted in low compound hit rates in robustness screening, where hit rates from a simulated screen were 1.0% (antagonist screen) and 0.1% (agonist screen). No continuous cell culture is required for the assay, reducing cell culture overheads and allowing the screen to run every day. Automation is simple and requires no temperature- or humidity-controlled incubation. No radioactivity is required. The method relies on measurement of cyclic AMP (cAMP) generation by fluorescence polarization assay using commercially available reagents. Membranes (1-2 microg protein per well, containing anti-cAMP antibody) are transferred to 384-well plates containing 1 microl test compound. For antagonist screens, agonist is added 15 min later. After 30 min incubation at room temperature, one further assay reagent (fluorescein-cAMP in a buffer containing detergent) is added. The signal may be read after 1 h and is stable for greater than 12 h. Typical Z' for the assay is approximately 0.5.     

A generic hierarchical screening method for the analysis of microscale refolds using an automated robotic platform

The refolding of protein derived from inclusion bodies is often characterized by low yields of active protein. The optimization of the refolding step is achieved empirically and consequently is time-consuming slowing process development. An automated robotic platform has been used to develop a dilution refold process-screening platform upon which a hierarchical set of assays rapidly determine optimal refolding conditions at the microscale. This hierarchy allows the simplest, cheapest, and most generic high-throughput assays to first screen for a smaller subset of potentially high-yielding conditions to take forward for analysis by slower, more expensive, or protein specific assays, thus saving resources whilst maximizing information output. An absorbance assay was used to initially screen out aggregating conditions, followed by an intrinsic fluorescence assay of the soluble protein to identify the presence of native-like tertiary structure, which was then confirmed by an activity assay. Results show that fluorescence can be used in conjunction with absorbance to eliminate low-yielding conditions, leaving a significantly reduced set of conditions from which the highest yielding ones can then be identified with slower and often more costly activity or RP-HPLC assays, thus reducing bottlenecks in high-throughput analysis. The microwell-based automated process sequence with generic hierarchical assays was also used to study and minimize the effect on redox potential or misfolding, of oxygenation due to agitation, before demonstrating that the platform can be used to rapidly collect data and evaluate different refolding conditions to speed up the acquisition of process development data in a resource efficient manner.     

Sensitive nonradioactive screening method for compounds interacting with alpha7-cholinoreceptor

A sensitive nonradioactive method for detection of substances interacting with the neuronal nicotinic acetylcholine alpha 7-type receptor (AChR) was proposed. The method uses biotinylated alpha-cobratoxin (Bt-CTX) and is based on the ability of the N-terminal ligand-binding extracellular domain (LBED) of AChR to interact with alpha-cobratoxin (CTX) as does the whole receptor. LBED was produced by heterologic expression of a gene fragment of the alpha 7 subunit of AChR from the rat brain in Escherichia coli cells sorbed on wells of a 96-well plate and incubated with Bt-CTX. The specifically bound Bt-CTX was determined by staining with streptavidin-peroxidase complex. The ability of other compounds to interact with alpha 7-AChR was checked according to the degree with which they inhibit Bt-CTX binding to LBED. Nicotine, carbamylcholine, d-tubocurarin, anabaseine, conotoxin ImI, and neurotoxin II were used as model compounds. The sensitivity of this method was comparable with that of the radioligand method (up to 10 pmol).     

A new high-throughput screening method for determining active and enantioselective hydrolases

A new high-throughput screening method using fluorescein sodium salt as an indicator to obtain hydrolases with high enantioselectivity is developed, which is demonstrated to be sensitive and reliable. The results determined by the method correlate well with those from GC analysis. This method can be applied to determine activity and enantioselectivity of not only lipase and esterase, but also other enzymes which catalyze hydrolysis reaction releasing proton, such as the protease or amidase. Because of the application of small amount of optically pure enantiomers, screening large libraries of enzymes is allowed at low cost and in short time.     

A sensitive colorimetric high-throughput screening method for lipase synthetic activity assay

A sensitive and practical high-throughput screening method for assaying lipase synthetic activity is described. Lipase-catalyzed transesterification between vinyl acetate and n-butanol in n-hexane was chosen as a model reaction. The released acetaldehyde was determined by the colorimetric method using 3-methyl-2-benzothialinone (MBTH) derivatization. In comparison with other methods, the major advantages of this process include high sensitivity, simple detection, inexpensive reagents, and low requirements for instruments.