Development and validation of a platelet calcium flux assay using a fluorescent imaging plate reader

Calcium signaling in platelets is an important physiological response to various aggregation stimuli. Loading platelets with various fluorescent dyes and measuring the change in calcium concentration using a spectrofluorometer has been the traditional approach to studying calcium signaling. This method suffers from the need for large platelet samples and a decrease in total fluorescence signal with time due to photobleaching. Therefore, it is rarely used to measure the quantitative effect of an agonist or antagonist on calcium signaling. Adaptation of these measurements to a fluorescent imaging plate reader (FLIPR) format allows the sample size to be reduced by 5- to 10-fold, and the microplate format allows a significant increase in throughput. Addition of the agonists to all wells simultaneously serves to normalize the total response. This article describes the first use of a FLIPR to study the calcium flux in human platelets. The IC(50) values showed a linear correlation with the K(i) for receptor binding in washed platelets. The generality of the methodology was shown by measuring EC(50) values for agonists and IC(50) values for antagonists of the platelet G protein-coupled receptor P2Y(1) and for the ion channel P2X(1).     

A bioluminescence assay for DNA methyltransferase activity based on methylation-resistant cleavage

DNA methyltransferase (MTase) is a kind of important regulatory factor in various biological processes. Current methods to investigate DNA MTase activity are still limited in the sensitivity and/or generality. Therefore, developing methods with high sensitivity and improved generality is needed. Here, we develop a new bioluminescence strategy based on methylation-resistant cleavage and protein expression in vitro to detect DNA MTase activity. In the strategy, Dam MTase was used as a model enzyme and MboI as the methylation-resistant endonuclease, and luciferase reporter DNA (LR-DNA) was used as their action target. Because the completely methylated LR-DNA could be expressed as detectable luciferase, Dam MTase activity was quantified by measuring the luminescence intensity of the expressed luciferase. The assay provides a very low detection limit (0.08 U/ml) as well as a wide linear range (0.2-100 U/ml). Besides, the analysis mode has improved generality and could be extended to the detection of other DNA MTases and the corresponding inhibitor screening.     

Development and validation of a fluorogenic assay to measure separase enzyme activity

Separase, an endopeptidase, plays a pivotal role in the separation of sister chromatids at anaphase by cleaving its substrate cohesin Rad21. Recent study suggests that separase is an oncogene. Overexpression of separase induces aneuploidy and mammary tumorigenesis in mice. Separase is also overexpressed and mislocalized in a wide range of human cancers, including breast, prostate, and osteosarcoma. Currently, there is no quantitative assay to measure separase enzymatic activity. To quantify separase enzymatic activity, we have designed a fluorogenic assay in which 7-amido-4-methyl coumaric acid (AMC)-conjugated Rad21 mitotic cleavage site peptide (Ac-Asp-Arg-Glu-Ile-Nle-Arg-MCA) is used as the substrate of separase. We used this assay to quantify separase activity during cell cycle progression and in a panel of human tumor cell lines as well as leukemia patient samples.     

Development and use of the assay for Legionella pneumophila detection based on fluorescent real-time/endpoint polymerase-chain reaction

The aim of the work was to develop a PCR-based assay for detection of L. pneumophila and L. micdadei in environmental samples as well as in clinical samples from low respiratory tract and to assess its analytic characteristics. The assay was used during investigation of the outbreak developed in July 2007 in town Verkhnyaya Pyshma (Sverdlovsk region). Polymerase-chain reaction (PCR)with fluorescent detection,sequencing and cloning of DNA fragments were used. Developed assay based on the PCR with fluorescent real-time/ endpointdetection is able to detect L. pneumophila in clinical and environmental samples and to quantify amount of bacterial DNA in water. Specificity of analysis (100%) was assessed using the panel of bacterial strains and samples from healthy individuals. Analytic sensitivity of assay and quantitation limit was 1000 GU in 1 ml. Sensitivity of the assay of artificially contaminated biological samples was 1000 bacteria in 1 ml. During outbreak investigation L. pneumophila DNAwas detected in 4 lung samples from 4 fatal cases, from 1 of 2 sputum samples, 1 of 2 bronchoalveolar lavage samples with X-ray confirmed pneumonia. Legionella's DNA was found in samples from cooling towers, central hot water supply as well as from showerheads in apartments of 3 patients. Fountain and drinking water samples were PCR-negative. Specificity of PCR-positive results was confirmed by sequencing. Use of the assay during outbreak in- vestigation allowed to confirm the diagnosis in fatal cases and quickly identify the possible source of infection.     

Development and validation of a generic 3D model of the distal femur

The development and validation of a virtual generic 3D model of the distal femur using computer graphical methods is presented. The synthesis of the generic model requires the following steps: acquisition of bony 3D morphology using standard computed tomography (CT) imaging; alignment of 3D models reconstructed from CT images with a common coordinate system; computer graphical sectioning of the models; extraction of bone contours from the image sections; combining and averaging of extracted contours; and 3D reconstruction of the averaged contours. The generic models reconstructed from the averaged contours of six cadaver femora were validated by comparing their surface geometry on a point to point basis with that of the CT reconstructed reference models. The mean errors ranged from 0.99 to 2.5 mm and were in agreement with the qualitative assessment of the models.     

Development and validation of a generic 3D model of the distal femur

The development and validation of a virtual generic 3D model of the distal femur using computer graphical methods is presented. The synthesis of the generic model requires the following steps: acquisition of bony 3D morphology using standard computed tomography (CT) imaging; alignment of 3D models reconstructed from CT images with a common coordinate system; computer graphical sectioning of the models; extraction of bone contours from the image sections; combining and averaging of extracted contours; and 3D reconstruction of the averaged contours.

The generic models reconstructed from the averaged contours of six cadaver femora were validated by comparing their surface geometry on a point to point basis with that of the CT reconstructed reference models. The mean errors ranged from 0.99 to 2.5 mm and were in agreement with the qualitative assessment of the models.     

A simple ultrasensitive method for the assay of cyclic AMP and cyclic GMP in tissues.

A simple and rapid method for a highly sensitive radioimmunoassay of cyclic AMP and GMP is described. The method is based on the observation that the affinity of the cyclic nucleotide antibodies for the 2′-0-succinyl or acetyl derivatives is considerably greater than that for the nonacylated cyclic nucleotides. With the present method, 3–10 fmoles of cyclic AMP and cyclic GMP can be assayed using commercially available antisera against cyclic AMP or cyclic GMP. A reproducible conversion of the cyclic nucleotides in aqueous samples to the 2′-0-acylated derivative is brought about by a simple one-step addition of premixed reagents containing either succinic anhydride or acetic anhydride and triethylamine. The time required for succinylation or acetylation of 100 samples is less than 5 min using this technique. Tissue extracts after purification through anion exchange resins do not interfere with the acylation. After acylation, labeled antigen and antiserum are added and incubated at 4°C for 15 hr. The bound antingen is separated from the unbound antigen by a simple ethanol precipitation using bovine serum albumin to ensure complete precipitation. The specificity of the method has been validated by the following criteria: (1) treatment of the samples with purified phosphodiesterase results in complete loss of cyclic nucleotide immunoreactivity, (2) cyclic nucleotide content was a linear function of the tissue weight, and (3) in one instance using bovine rod outer segments, identical values of cyclic GMP were obtained with the present method and a purely physical method (high pressure liquid chromatography).     

Development of a generic dual-reporter gene assay for screening G-protein-coupled receptors

Multiple assay formats have been developed for the pharmacological characterization of G-protein-coupled receptors (GPCRs) and for screening orphan receptors. However, the increased pace of target identification and the rapid expansion of compound libraries present the need to develop novel assay formats capable of screening multiple GPCRs simultaneously. To address this need, the authors have developed a generic dual-reporter gene assay that can detect ligand activity at 2 GPCRs within the same assay. Two stable HEK293 cell lines were generated expressing either a firefly (Photinus) luciferase gene under the control of multiple cAMP-response elements (CREs) or a Renilla luciferase gene under the control of multiple 12-O-tetradecanoylphorbol-13-acetate (TPA)-responsive elements (TREs). Coseeded reporter cells were used to assess ligand binding activity at both Galphas-and Galphaq-coupled receptors. By selectively coexpressing receptors with a chimeric G-protein, agonist activity was assessed at Galphai/o-coupled receptors in combination with either Galphas-or Galphaq-coupled receptors. The dual-reporter gene assay was shown to be capable of simultaneously performing duplexed screens for a variety of agonist and/or antagonist combinations. The data generated from the duplexed reporter assays were pharmacologically relevant, and Z' factor analysis indicated the suitability of both agonist and antagonist screens for use in high-throughput screening.     

Development and use of a vaccinia virus neutralization assay based on flow cytometric detection of green fluorescent protein

A rapid and sensitive neutralization assay is required to evaluate alternative smallpox vaccines. Here we describe the development and use of a 96-well plate, semi-automated, flow cytometric assay that uses a recombinant vaccinia virus expressing enhanced green fluorescent protein and which would be applicable to other viruses.     

Development of a fluorescent ligand-binding assay using the AcroWell filter plate

One of the most powerful tools for receptor research and drug discovery is the use of receptor-ligand affinity screening of combinatorial libraries. Early work involved the use of radioactive ligands to identify a binding event; however, there are numerous limitations involved in the use of radioactivity for high throughput screening. These limitations have led to the creation of highly sensitive, nonradioactive alternatives to investigate receptor-ligand interactions. Pall Gelman Laboratory has introduced the AcroWell, a patented low-fluorescent-background membrane and sealing process together with a filter plate design that is compatible with robotic systems. Taken together, these allow the AcroWell 96-well filter plate to detect trace quantities of lanthanide-labeled ligands for cell-, bead-, or membrane-based assays using time-resolved fluorescence. Using europium-labeled galanin, we have demonstrated that saturation binding experiments can be performed with low-background fluorescence and signal-to-noise ratios that rival traditional radioisotopic techniques while maintaining biological integrity of the receptor-ligand interaction. In addition, the ability to discriminate between active and inactive compounds in a mock galanin screen is demonstrated with low well-to-well variability, allowing reliable determination of positive hits even for low-affinity interactions.     

A general liquid chromatography/mass spectroscopy-based assay for detection and quantitation of methyltransferase activity

Methyltransferases form a large class of enzymes, most of which use S-adenosylmethionine as the methyl donor. In fact, S-adenosylmethionine is second only to ATP in the variety of reactions for which it serves as a cofactor. Several methods to measure methyltransferase activity have been described, most of which are applicable only to specific enzymes and/or substrates. In this work we describe a sensitive liquid chromatography/mass spectroscopy-based methyltransferase assay. The assay monitors the conversion of S-adenosylmethionine to S-adenosylhomocysteine and can be applied to any methyltransferase and substrate of interest. We used the well-characterized enzyme catechol O-methyltransferase to demonstrate that the assay can monitor activity with a variety of substrates, can identify new substrates, and can be used even with crude preparation of enzyme. Furthermore, we demonstrate the utility of the assay for kinetic characterization of enzymatic activity.     

Development and validation of an immunoreceptor assay for simulect based on surface plasmon resonance.

Simulect is a chimeric human/mouse antibody directed against interleukin-2 (IL-2) receptor. A combined immuno- and receptor assay has been developed and validated to characterize the production of Simulect batches. This assay is based on surface plasmon resonance (SPR) technology. In each experiment two successive interactions were monitored: the direct binding of Simulect to an anti-human IgG antibody, followed by the direct binding of IL-2-soluble receptor to the preformed anti-human IgG antibody/Simulect complex. Based on the first interaction a direct immunoassay for Simulect was optimized and validated. Based on the second interaction a direct receptor assay for Simulect biological activity was optimized and validated. The assays were validated by performing three independent assays on 3 different days. The intra- and interday variations of the immunoassay (expressed as % CV) were, respectively, 1.7 and 1.6%. The overall accuracy for the immunoassay was 98.5% +/- 1. The intra- and interday variations of the receptor assay (% CV) were, respectively, 1.6 and 3.7%. The overall accuracy of the receptor assay was 100% +/- 2. Four batches of Simulect were compared to a reference batch. The results did not show significant differences for the immunoreactivity. However, the results of the receptor assay showed accuracies which were apparently higher than 100%. This was explained by a slight degradation of the reference batch after few years of storage. These results demonstrate the advantage of this method combining evaluation of the immunological and biological integrity of the drug and a high reproducibility in accuracy and precision of the biosensor-based technology.     

DNA methyltransferase inhibitor assay system based on the HBx-induced DNA methylation of E-cadherin

We here report a simple assay system for DNA methyltransferase (DNMT) inhibitors based on the HBx-induced DNA methylation of E-cadherin. A stable cell line named G1 was generated by co-transfecting E-cadherin luciferase reporter and HBx-expression plasmid into HepG2 cells. Treatment of G1 cells with DNMT inhibitors, 5-azacytidine, 5-aza-2′-deoxycytidine, and procainamaid, dose-dependently inhibited DNA methylation of E-cadherin promoter in the reporter, resulting in up-regulation of luciferase levels and its enzyme activity. Treatment with all-trans retinoic acid that is known to inhibit DNMT expression, also induced similar effects. Our system can be useful for development of epi-drugs targeting DNA methylation in malignancies.     

Ratiometric assay of CARM1 activity using a FRET-based fluorescent probe

One of the regulatory mechanisms of epigenetic gene expression is the post-translational methylation of arginine residues, which is catalyzed by protein arginine methyltransferases (PRMTs). Abnormal expression of PRMT4/CARM1, one of the PRMTs, is associated with various diseases, including cancers. Here, we designed and synthesized a Förster resonance energy transfer (FRET)-based probe, FRC, which contains coumarin and fluorescein fluorophores at the N-terminus and C-terminus of a peptide containing an arginine residue within an appropriate amino acid sequence to serve as a substrate of CARM1; the two fluorophores act as a FRET donor and a FRET acceptor, respectively. Since trypsin specifically hydrolyzes the arginine residue, but not a monomethylarginine or dimethylarginine residue, CARM1 activity can be evaluated from the change of the coumarin/fluorescein fluorescence ratio of FRC in the presence of trypsin.     

Determination of lipoprotein lipase activity using a novel fluorescent lipase assay

A novel, real-time, homogeneous fluorogenic lipoprotein lipase (LPL) assay was developed using a commercially available substrate, the EnzChek lipase substrate, which is solubilized in Zwittergent. The triglyceride analog substrate does not fluoresce, owing to apposition of fluorescent and fluorescent quenching groups at the sn-1 and sn-2 positions, respectively, fluorescence becoming unquenched upon release of the sn-1 BODIPY FA derivative following hydrolysis. Increase in fluorescence intensity at 37°C was proportional to LPL concentration. The assay was more sensitive than a similar assay using 1,2-O-dilauryl-rac-glycero-3-glutaric acid-(6-methylresorufin ester) and was validated in biological samples, including determination of LPL-specific activity in postheparin mouse plasma. The simplicity and reproducibility of the assay make it ideal for in vitro, high-throughput screening for inhibitors and activators of LPL, thus expediting discovery of drugs of potential clinical value.     

A versatile non-radioactive assay for DNA methyltransferase activity and DNA binding

We present a simple, non-radioactive assay for DNA methyltransferase activity and DNA binding. As most proteins are studied as GFP fusions in living cells, we used a GFP binding nanobody coupled to agarose beads (GFP nanotrap) for rapid one-step purification. Immobilized GFP fusion proteins were subsequently incubated with different fluorescently labeled DNA substrates. The absolute amounts and molar ratios of GFP fusion proteins and bound DNA substrates were determined by fluorescence spectroscopy. In addition to specific DNA binding of GFP fusion proteins, the enzymatic activity of DNA methyltransferases can also be determined by using suicide DNA substrates. These substrates contain the mechanism-based inhibitor 5-aza-dC and lead to irreversible covalent complex formation. We obtained covalent complexes with mammalian DNA methyltransferase 1 (Dnmt1), which were resistant to competition with non-labeled canonical DNA substrates, allowing differentiation between methyltransferase activity and DNA binding. By comparison, the Dnmt1^C1229W catalytic site mutant showed DNA-binding activity, but no irreversible covalent complex formation. With this assay, we could also confirm the preference of Dnmt1 for hemimethylated CpG sequences. The rapid optical read-out in a multi-well format and the possibility to test several different substrates in direct competition allow rapid characterization of sequence-specific binding and enzymatic activity.     

Development and validation of a simple cell-based fluorescence assay for dipeptidyl peptidase 1 (DPP1) activity

Dipeptidyl peptidase 1 (DPP1) (EC 3.4.14.1; also known as cathepsin C, cathepsin J, dipeptidyl aminopeptidase, and dipeptidyl aminotransferase) is a lysosomal cysteinyl protease of the papain family involved in the intracellular degradation of proteins. Isolated enzyme assays for DPP1 activity using a variety of synthetic substrates such as dipeptide or peptide linked to amino-methyl-coumarin (AMC) or other fluorophores are well established. There is, however, no report of a simple whole-cell-based assay for measuring lysosomal DPP1 activity other than the use of flow cytometry (fluorescence-activated cell sorting) or the use of invasive activity-based probes or the production of physiological products such as neutrophil elastase. The authors investigated a number of DPP1 fluorogenic substrates that have the potential to access the lysosome and enable the measurement of DPP1 enzyme activity in situ. They describe the development and evaluation of a simple noninvasive fluorescence assay for measuring DPP1 activity in fresh or cryopreserved human THP-1 cells using the substrate H-Gly-Phe-AFC (amino-fluoro-coumarin). This cell-based fluorescence assay can be performed in a 96-well plate format and is ideally suited for determining the cell potency of potential DPP1 enzyme inhibitors.     

Development and validation of a cytochrome c-coupled assay for pteridine reductase 1 and dihydrofolate reductase

Activity of the pterin- and folate-salvaging enzymes pteridine reductase 1 (PTR1) and dihydrofolate reductase-thymidylate synthetase (DHFR-TS) is commonly measured as a decrease in absorbance at 340 nm, corresponding to oxidation of nicotinamide adenine dinucleotide phosphate (NADPH). Although this assay has been adequate to study the biology of these enzymes, it is not amenable to support any degree of routine inhibitor assessment because its restricted linearity is incompatible with enhanced throughput microtiter plate screening. In this article, we report the development and validation of a nonenzymatically coupled screening assay in which the product of the enzymatic reaction reduces cytochrome c, causing an increase in absorbance at 550 nm. We demonstrate this assay to be robust and accurate, and we describe its utility in supporting a structure-based design, small-molecule inhibitor campaign against Trypanosoma brucei PTR1 and DHFR-TS.