Mapping the functionality of GTPases through small molecule inhibitors represents an underexplored area in large part due to the lack of suitable compounds. Here we report on the small chemical molecule 2-(benzoylcarbamothioylamino)-5,5-dimethyl-4,7-dihydrothieno[2,3-c]pyran-3-carboxylic acid (PubChem CID 1067700) as an inhibitor of nucleotide binding by Ras-related GTPases. The mechanism of action of this pan-GTPase inhibitor was characterized in the context of the Rab7 GTPase as there are no known inhibitors of Rab GTPases. Bead-based flow cytometry established that CID 1067700 has significant inhibitory potency on Rab7 nucleotide binding with nanomolar inhibitor (Ki) values and an inhibitory response of ≥97% for BODIPY-GTP and BODIPY-GDP binding. Other tested GTPases exhibited significantly lower responses. The compound behaves as a competitive inhibitor of Rab7 nucleotide binding based on both equilibrium binding and dissociation assays. Molecular docking analyses are compatible with CID 1067700 fitting into the nucleotide binding pocket of the GTP-conformer of Rab7. On the GDP-conformer, the molecule has greater solvent exposure and significantly less protein interaction relative to GDP, offering a molecular rationale for the experimental results. Structural features pertinent to CID 1067700 inhibitory activity have been identified through initial structure activity analyses and identified a molecular scaffold that may serve in the generation of more selective probes for Rab7 and other GTPases. Taken together, our study has identified the first competitive GTPase inhibitor and demonstrated the potential utility of the compound for dissecting the enzymology of the Rab7 GTPase as well as serving as a model for other small molecular weight GTPase inhibitors.
Rab, Rho, Rac, Cdc42 and Ras GTPases; chemical biology; drug discovery; therapeutics; fluorescent GTP and GDP; enzyme kinetics
A new multiplexed, bead-based method which utilizes nucleic acid hybridizations on the surface of microscopic polystyrene spheres to identify specific sequences in heterogeneous mixtures of DNA sequences is described. The method consists of three elements: beads (5.6-μm diameter) with oligomer capture probes attached to the surface, three fluorophores for multiplexed detection, and flow cytometry instrumentation. Two fluorophores are impregnated within each bead in varying amounts to create different bead types, each associated with a unique probe. The third fluorophore is a reporter. Following capture of fluorescent cDNA sequences from environmental samples, the beads are analyzed by flow cytometric techniques which yield a signal intensity for each capture probe proportional to the amount of target sequences in the analyte. In this study, a direct hybrid capture assay was developed and evaluated with regard to sequence discrimination and quantitation of abundances. The target sequences (628 to 728 bp in length) were obtained from the 16S/23S intergenic spacer region of microorganisms collected from polluted groundwater at the nuclear waste site in Hanford, Wash. A fluorescence standard consisting of beads with a known number of fluorescent DNA molecules on the surface was developed, and the resolution, sensitivity, and lower detection limit for measuring abundances were determined. The results were compared with those of a DNA microarray using the same sequences. The bead method exhibited far superior sequence discrimination and possesses features which facilitate accurate quantitation.
Micron scale latex beads are well established as highly biocompatible reagents. Imbibing two fluorescent dyes into the interior of the beads enables the creation of a family of combinatorially colored labels. Previous use of such beads, in flow cytometry for example, has focused on beads of ~5μm diameter. We show here that 280 nm combinatorially labeled particles can be used to create ELISA-style assays in 200 μm scale virtual wells, using digital microscopy as the readout. The utility of this technique is illustrated by profiling the secreted cytokine footprints of peripheral blood mononuclear cells in a multiparametric version of the popular Elispot assay. Doing so reveals noncanonical classes of T lymphocytes. We further show that the secreting cell type can be concurrently identified by surface staining with a cell type specific antibody conjugated to the same multiplexed beads.
cytokines; lymphocytes; multiplex; high content assay
The Bio-Plex® system utilizes xMAP technology to permit the multiplexing of up to 100 different analytes. Multiplex analysis gives researchers the ability to look at analytes simultaneously providing more information from less sample volume in less time than traditional immunoassay methods. Similar to ELISA, xMAP utilizes an antibody sandwich for detection but differs from ELISA in capture substrate and detection method. Rather than a flat surface, Bio-Plex®assays make use of differentially detectable bead sets as a substrate capturing analytes in solution and employs fluorescent methods for detection. These bead sets identify the analytes and detection antibodies are used to measure the quantity of analyte. The use of differentially detectable beads enables the simultaneous identification and quantification of many analytes in the same sample.
Immunoassay; multiplex detection; ELISA; Bio-Plex®; xMAP; Luminex
Ras-like small GTPases cycle between GTP-bound active and GDP-bound inactive conformational states to regulate diverse cellular processes. Despite their importance, detailed kinetic or comparative studies of family members are rarely undertaken due to the lack of real-time assays measuring nucleotide binding or exchange. Here, we report a bead-based, flow cytometric assay that quantitatively measures the nucleotide binding properties of GST-chimeras for prototypical Ras-family members Rab7 and Rho. Measurements are possible in the presence or absence of Mg2+, with magnesium cations principally increasing affinity and slowing nucleotide dissociation rate 8- to 10-fold. GST-Rab7 exhibited a 3-fold higher affinity for GDP relative to GTP that is consistent with a 3-fold slower dissociation rate of GDP. Strikingly, GST-Rab7 had a marked preference for GTP with ribose ring-conjugated BODIPY FL. The more commonly used γ-NH-conjugated BODIPY FL GTP analogue failed to bind to GST-Rab7. In contrast, both BODIPY analogues bound equally well to GST-RhoA and GST-RhoC. Comparisons of the GST-Rab7 and GST-RhoA GTP-binding pockets provide a structural basis for the observed binding differences. In sum, the flow cytometric assay can be used to measure nucleotide binding properties of GTPases in real-time and quantitatively assess differences between GTPases.
Rab and Rho GTPases; membrane trafficking; actin remodeling; nucleotide binding and exchange; fluorescent GTP analogues
Efficient and reliable diagnostic tools for the routine indexing and certification of clean propagating material are essential for the management of pospiviroid diseases in horticultural crops. This study describes the development of a true multiplexed diagnostic method for the detection and identification of all nine currently recognized pospiviroid species in one assay using Luminex bead-based suspension array technology. In addition, a new data-driven, statistical method is presented for establishing thresholds for positivity for individual assays within multiplexed arrays. When applied to the multiplexed array data generated in this study, the new method was shown to have better control of false positives and false negative results than two other commonly used approaches for setting thresholds. The 11-plex Luminex MagPlex-TAG pospiviroid array described here has a unique hierarchical assay design, incorporating a near-universal assay in addition to nine species-specific assays, and a co-amplified plant internal control assay for quality assurance purposes. All assays of the multiplexed array were shown to be 100% specific, sensitive and reproducible. The multiplexed array described herein is robust, easy to use, displays unambiguous results and has strong potential for use in routine pospiviroid indexing to improve disease management strategies.
Cytokines and chemokines are relevant biomarkers of pathology and immunity to infectious diseases such as malaria. Several commercially available kits based on quantitative suspension array technologies allow the profiling of multiple cytokines and chemokines in small volumes of sample. However, kits are being continuously improved and information on their performance is lacking.
Different cytokine/chemokine kits, two flow cytometry-based (eBioscience® FlowCytomix™ and BD™ Cytometric Bead Array Human Enhanced Sensitivity) and four Luminex®-based (Invitrogen™ Human Cytokine 25-Plex Panel, Invitrogen™ Human Cytokine Magnetic 30-Plex Panel, Bio-Rad® Bio-Plex Pro™ Human Cytokine Plex Assay and Millipore™ MILLIPLEX® MAP Plex Kit) were compared. Samples tested were supernatants of peripheral blood mononuclear cells of malaria-exposed children stimulated with Plasmodium falciparum parasite lysates. Number of responses in range that could be detected was determined and reproducibility of duplicates was evaluated by the Bland-Altman test. Luminex® kits performed better than flow cytometry kits in number of responses in range and reproducibility. Luminex® kits were more reproducible when magnetic beads were used. However, within each methodology overall performance depended on the analyte tested in each kit. Within the Luminex® kits, the Invitrogen™ with polystyrene beads had the poorer performance, whereas Invitrogen™ with magnetic beads had the higher percentage of cytokines/chemokines with both readings in range (40%), followed by Bio-Rad® with magnetic beads (35%). Regarding reproducibility, the Millipore™ kit had the highest percentage (60%) of cytokines/chemokines with acceptable limits of agreement (<30%), followed by the Invitrogen™ with magnetic beads (40%) that had tighter limits of agreement.
Currently available kits for cytokine and chemokine quantification differ in reproducibility and concentration range of accurate detection. Luminex®-based kits with magnetic beads perform the best. Data highlights the importance of testing different kits before each study to choose the most appropriate, depending on the priority of the cytokines assessed.
This review describes the use of high-throughput flow cytometry for performing multiplexed cell-based and bead-based screens. With the many advances in cell-based analysis and screening, flow cytometry has historically been underutilized as a screening tool largely due to the limitations in handling large numbers of samples. However, there has been a resurgence in the use of flow cytometry due to a combination of innovations around instrumentation and a growing need for cell-based and bead-based applications. The HTFC™ Screening System (IntelliCyt Corporation, Albuquerque, NM) is a novel flow cytometry-based screening platform that incorporates a fast sample-loading technology, HyperCyt®, with a two-laser, six-parameter flow cytometer and powerful data analysis capabilities. The system is capable of running multiplexed screening assays at speeds of up to 40 wells per minute, enabling the processing of a 96- and 384-well plates in as little as 3 and 12 min, respectively. Embedded in the system is HyperView®, a data analysis software package that allows rapid identification of hits from multiplexed high-throughput flow cytometry screening campaigns. In addition, the software is incorporated into a server-based data management platform that enables seamless data accessibility and collaboration across multiple sites. High-throughput flow cytometry using the HyperCyt technology has been applied to numerous assay areas and screening campaigns, including efflux transporters, whole cell and receptor binding assays, functional G-protein-coupled receptor screening, in vitro toxicology, and antibody screening.
We have identified a human Rho protein, RhoE, which has unusual structural and biochemical properties that suggest a novel mechanism of regulation. Within a region that is highly conserved among small GTPases, RhoE contains amino acid differences specifically at three positions that confer oncogenicity to Ras (12, 59, and 61). As predicted by these substitutions, which impair GTP hydrolysis in Ras, RhoE binds GTP but lacks intrinsic GTPase activity and is resistant to Rho-specific GTPase-activating proteins. Replacing all three positions in RhoE with conventional amino acids completely restores GTPase activity. In vivo, RhoE is found exclusively in the GTP-bound form, suggesting that unlike previously characterized small GTPases, RhoE may be normally maintained in an activated state. Thus, amino acid changes in Ras that are selected during tumorigenesis have evolved naturally in this Rho protein and have similar consequences for catalytic function. All previously described Rho family proteins are modified by geranylgeranylation, a lipid attachment required for proper membrane localization. In contrast, the carboxy-terminal sequence of RhoE predicts that, like Ras proteins, RhoE is normally farnesylated. Indeed, we have found that RhoE in farnesylated in vivo and that this modification is required for association with the plasma membrane and with an unidentified cellular structure that may play a role in adhesion. Thus, two unusual structural features of this novel Rho protein suggest a striking evolutionary divergence from the Rho family of GTPases.
Given their medical importance, proteases have been studied by diverse approaches and screened for small molecule protease inhibitors. Here, we present a multiplexed microsphere-based protease assay that uses high-throughput flow cytometry to screen for inhibitors of the light chain protease of botulinum neurotoxin type A (BoNTALC). Our assay uses a full-length substrate and several deletion mutants screened in parallel to identify small molecule inhibitors. The use of multiplex flow cytometry has the advantage of using full-length substrates, which contain already identified distal-binding elements for the BoNTALC, and could lead to a new class of BoNTALC inhibitors. In this study, we have screened 880 off patent drugs and bioavailable compounds to identify ebselen as an in vitro inhibitor of BoNTALC. This discovery demonstrates the validity of our microsphere-based approach and illustrates its potential for high-throughput screening for inhibitors of proteases in general.
The need for a rapid and accurate method for the detection of fungal pathogens has become imperative as the incidence of fungal infections has increased dramatically. Herein, we tested the Luminex 100, a novel flow cytometer, for the detection of the medically important genus Trichosporon. This genus was selected as our proof-of-concept model due to the close phylogenetic relationship between the species. The method, which is based on a nucleotide hybridization assay, consists of a combination of different sets of fluorescent beads covalently bound to species-specific capture probes. Upon hybridization, the beads bearing the target amplicons are classified by their spectral addresses with a 635-nm laser. Quantitation of the hybridized biotinylated amplicon is based on fluorescence detection with a 532-nm laser. We tested in various multiplex formats 48 species-specific and group-specific capture probes designed in the D1/D2 region of ribosomal DNA, internal transcribed spacer regions, and intergenic spacer region. Species-specific biotinylated amplicons were generated with three sets of primers to yield fragments from the three regions. The assay was specific and fast, as it discriminated species differing by 1 nucleotide and required less than 50 min following amplification to process a 96-well plate. The sensitivity of the assay allowed the detection of 102 genome molecules in PCRs and 107 to 108 molecules of biotinylated amplification product. This technology provided a rapid means of detection of Trichosporon species with the flexibility to identify species in a multiplex format by combining different sets of beads.
Before the era of tyrosine kinase inhibitors (TKIs), interferon-alpha (IFN-α) was the treatment of choice in chronic myeloid leukemia (CML). Curiously, some IFN-α treated patients were able to discontinue therapy without disease progression. The aim of this project was to study the immunomodulatory effects of IFN-α in CML patients in prolonged remission and isolate biological markers predicting response. Due to rarity of patients on IFN-α monotherapy, a relatively small cohort of patients still on treatment (IFN-ON, n = 10, median therapy duration 11.8 years) or had discontinued IFN-α therapy but remained in remission for >2 years (IFN-OFF, n = 9) were studied. The lymphocyte immunophenotype was analyzed with a comprehensive flow cytometry panel and plasma cytokine levels were measured with multiplex bead-based assay. In addition, the clonality status of different lymphocyte subpopulations was analyzed by TCR γ/δ rearrangement assay. Median NK-cell absolute number and proportion from lymphocytes in blood was higher in IFN-OFF patients as compared to IFN-ON patients or controls (0.42, 0.19, 0.21×109/L; 26%, 12%, 11%, respectively, p<0.001). The proportion of CD8+ T-cells was significantly increased in both patient groups and a larger proportion of T-cells expressed CD45RO. Most (95%) patients had significant numbers of oligoclonal lymphocytes characterized by T-cell receptor γ/δ rearrangements. Strikingly, in the majority of patients (79%) a distinct clonal Vγ9 gene rearrangement was observed residing in γδ+ T-cell population. Similar unique clonality pattern was not observed in TKI treated CML patients. Plasma eotaxin and MCP-1 cytokines were significantly increased in IFN-OFF patients. Despite the limited number of patients, our data indicates that IFN-α treated CML patients in remission have increased numbers of NK-cells and clonal γδ+ T-cells and a unique plasma cytokine profile. These factors may relate to anti-leukemic effects of IFN-α in this specific group of patients and account for prolonged therapy responses even after drug discontinuation.
This study was conducted to examine the frequency, phenotype, and functional profile of T lymphocytes that proliferate in response to type I collagen (CI) in patients with scleroderma (SSc). Peripheral blood mononuclear cells (PBMCs) from SSc patients, healthy controls, and rheumatoid arthritis disease controls were labeled with carboxy-fluorescein diacetate, succinimidyl ester (CFSE), cultured with or without antigen (bovine CI) for 14 days, and analysed by flow cytometry. Surface markers of proliferating cells were identified by multi-color flow cytometry. T-cell lines were derived after sorting for proliferating T cells (CFSElow). Cytokine expression in CI-responsive T cells was detected by intracellular staining/flow cytometry and by multiplex cytokine bead assay (Bio-Plex). A T-cell proliferative response to CI was detected in 8 of 25 (32%) SSc patients, but was infrequent in healthy or disease controls (3.6%; p = 0.009). The proliferating T cells expressed a CD4+, activated (CD25+), memory (CD45RO+) phenotype. Proliferation to CI did not correlate with disease duration or extent of skin involvement. T-cell lines were generated using in vitro CI stimulation to study the functional profile of these cells. Following activation of CI-reactive T cells, we detected intracellular interferon (IFN)-γ but not interleukin (IL)-4 by flow cytometry. Supernatants from the T-cell lines generated in vitro contained IL-2, IFN-γ, GM-CSF (granulocyte macrophage-colony-stimulating factor), and tumour necrosis factor-α, but little or no IL-4 and IL-10, suggesting that CI-responsive T cells express a predominantly Th1 cytokine pattern. In conclusion, circulating memory CD4 T cells that proliferate to CI are present in a subset of patients with SSc, but are infrequent in healthy or disease controls.
The recent introduction of fluorescent bead-based technology, allowing the measurement of multiples analytes in a single 25–50 µl sample has revolutionized the study of cytokine responses. However, such multiplex approaches may compromise the ability of these assays to accurately measure actual cytokine levels. This study evaluates the performance of three commercially available multiplex cytokine fluorescent bead-based immunoassays (Bio-Rad's Cytokine 17-plex kit; LINCO Inc's 29-plex kit; and RnD System's Fluorokine-Multi Analyte Profiling (MAP) base kit A and B). The LINCO Inc kit was found to be the most sensitive assay for measuring concentrations of multiple recombinant cytokines in samples that had been spiked with serial dilutions of the standard provided by the manufacturer, followed respectively by the RnD Fluorokine-(MAP) and Bio-Rad 17-plex kits. A positive correlation was found in the levels of IFN-γ measured in antigen stimulated whole blood culture supernatants by the LINCO Inc 29-plex, RnD Fluorokine-(MAP) and RnD system IFN-γ Quantikine ELISA kits across a panel of controls and stimulated samples. Researchers should take the limitation of such multiplexed assays into account when planning experiments and the most appropriate use for these tests may currently be as screening tools for the selection of promising markers for analysis by more sensitive techniques.
A method is described for the quantitative analysis of protein-protein interactions using the Flow Cytometry Protein Interaction Assay (FCPIA). This method is based upon immobilizing protein on a polystyrene bead, incubating these beads with a fluorescently labeled binding partner, and assessing the sample for bead-associated fluorescence in a flow cytometer. This method can be used to calculate protein-protein interaction affinities or to perform competition experiments with unlabeled binding partners or small molecules. Examples described in this protocol highlight the use of this assay in the quantification of the affinity of binding partners of the Regulator of G-Protein Signaling protein, RGS19, in either a saturation or competition format. An adaptation of this method that is compatible for High Throughput screening is also provided.
RGS; G protein; Protein-Protein Interaction; FCPIA; High Throughput Screening; Multiplexing
Coccidiostats are the only veterinary drugs still permitted to be used as feed additives to treat poultry for coccidiosis. To protect consumers, maximum levels for their presence in food and feed have been set by the European Union (EU). To monitor these coccidiostats, a rapid and inexpensive screening method would be a useful tool. The development of such a screening method, using a flow cytometry-based immunoassay, is described. The assay uses five sets of colour-coded paramagnetic microspheres for the detection of six selected priority coccidiostats. Different coccidiostats, with and without carrier proteins, were covalently coupled onto different bead sets and tested in combination with polyclonal antisera and with a fluorescent-labelled secondary antibody. The five optimal combinations were selected for this multiplex and a simple-to-use sample extraction method was applied for screening blank and spiked eggs and feed samples. A very good correlation (r ranging from 0.995 to 0.999) was obtained with the responses obtained in two different flow cytometers (Luminex 100 and FLEXMAP 3D). The sensitivities obtained were in accordance with the levels set by the EU as the measured limits of detection for narasin/salinomycin, lasalocid, diclazuril, nicarbazin (4,4′-dinitrocarbanilide) and monensin in eggs were 0.01, 0.1, 0.5, 53 and 0.1 μg/kg and in feed 0.1, 0.2, 0.3, 9 and 1.5 μg/kg, respectively.
Coccidiostats; Multiplex flow cytometric immunoassay; Colour-coded beads; Eggs; Feed
Chronic lung diseases are marked by progressive inflammation, tissue damage and remodelling. Bone marrow-derived progenitor cells may contribute to these processes. The objectives of this study were to (1) to quantify CD45+Collagen-1+ fibrocytes and a novel epithelial-like population of bone marrow-derived cells, which express Clara Cell Secretory Protein, in patients at the time of lung transplant and (2) to evaluate mediators that may act to recruit these cells during injury.
Using an observational design, progenitor cells were quantified by flow cytometry from both bone marrow (BM) and peripheral blood (PB). Migration was tested using in vitro transwell assays. Multiplex bead-based assays were used to quantify plasma cytokines.
An increase in CD45+Collagen-1+ fibrocytes was found in pulmonary fibrosis and bronchiolitis obliterans patients. Cystic fibrosis patients had an increase in CCSP+ cells in both the BM and PB. The proportion of CCSP+ cells in the BM and PB was correlated. CCSP+ cells express the chemokine receptors CCR2, CCR4, CXCR3, and CXCR4, and significantly migrated in vitro toward Stromal Derived Factor-1 (SDF-1) and Stem Cell Growth Factor-β (SCGF-β). Plasma cytokine levels differed between disease groups, with a significant correlation between SCGF-β and CCSP+ cells and between Monocyte Chemotactic Protein-1 and fibrocytes.
Different bone marrow-derived cells are found in various lung diseases. Increased fibrocytes were associated with fibrotic lung diseases. An increase in the novel CCSP+ epithelial-like progenitors in cystic fibrosis patients was found. These differences may be mediated by alterations in plasma cytokines responsible for cell recruitment.
Fibrocytes; Clara cell; Lung progenitor; Migration
Prostatic acid phosphatase (PAP) is expressed in nociceptive neurons and functions as an ectonucleotidase. Injection of the secretory isoform of PAP has potent antinociceptive effects in mouse models of chronic pain. These data suggested that a small molecule activator of PAP may have utility as a novel therapeutic for chronic pain, while inhibitors could be used to acutely inhibit PAP in vitro and in vivo. To identify small molecule modulators of PAP activity, we validated a high throughput, fluorescence-based biochemical assay and then used this assay to screen a compound library. We decreased the frequency of false positive activators by subtracting compound fluorescence from the final assay fluorescence. This approach significantly reduced the number of false positive activators found in the screen. While no activators were confirmed, seven novel inhibitors of PAP were identified. Our results suggest this high throughput assay could be used to identify small molecule modulators of PAP activity.
The small GTPase cycles between the inactive GDP form and the activated GTP form, catalyzed by the upstream guanine exchange factors. The modulation of such process by small molecules has been proven to be a fruitful route for therapeutic intervention to prevent the over-activation of the small GTPase. The fragment based approach emerging in the past decade has demonstrated its paramount potential in the discovery of inhibitors targeting such novel and challenging protein-protein interactions. The details regarding the procedure of NMR fragment screening from scratch have been rarely disclosed comprehensively, thus restricts its wider applications. To achieve a consistent screening applicable to a number of targets, we developed a highly automated protocol to cover every aspect of NMR fragment screening as possible, including the construction of small but diverse libray, determination of the aqueous solubility by NMR, grouping compounds with mutual dispersity to a cocktail, and the automated processing and visualization of the ligand based screening spectra. We exemplified our streamlined screening in RhoA alone and the complex of the small GTPase RhoA and its upstream guanine exchange factor LARG. Two hits were confirmed from the primary screening in cocktail and secondary screening over individual hits for LARG/RhoA complex, while one of them was also identified from the screening for RhoA alone. HSQC titration of the two hits over RhoA and LARG alone, respectively, identified one compound binding to RhoA.GDP at a 0.11 mM affinity, and perturbed the residues at the switch II region of RhoA. This hit blocked the formation of the LARG/RhoA complex, validated by the native gel electrophoresis, and the titration of RhoA to 15N labeled LARG in the absence and presence the compound, respectively. It therefore provides us a starting point toward a more potent inhibitor to RhoA activation catalyzed by LARG.
We established a homogeneous luminescent oxygen channeling sensor for measuring activation states of small GTPases. The assay quantifies activated GTPases in cell lysates, can be applied to different GTPases, and can be used for multiplex screening. The study will provide guidelines for determining activation states of diverse GTPases in various biological contexts.
Sample preparation for Roche/454, ABI/SOLiD and Life Technologies/Ion Torrent sequencing are based on amplification of library fragments on the surface of beads prior to sequencing. Commonly, libraries are barcoded and pooled, to maximise the sequence output of each sequence run. Here, we describe a novel approach for normalization of multiplex next generation sequencing libraries after emulsion PCR. Briefly, amplified libraries carrying unique barcodes are prepared by fluorescent tagging of complementary sequences and then resolved by high-speed flow cytometric sorting of labeled emulsion PCR beads. The protocol is simple and provides an even sequence distribution of multiplex libraries when sequencing the flow-sorted beads. Moreover, since many empty and mixed emulsion PCR beads are removed, the approach gives rise to a substantial increase in sequence quality and mean read length, as compared to that obtained by standard enrichment protocols.
The advent of multiplexed bead assays in recent years has introduced a new dimension of testing for complex diseases such as lupus, which can involve multiple autoantibodies. The ability to rapidly identify multiple autoantibodies, with high sensitivity and specificity in an automated fashion, is highly attractive. The aim of this study was to assess the performance and clinical value of multiplexed bead-based (AtheNA Multi-Lyte ANA-II test system) immunoassays both by comparing the results with those achieved by indirect fluorescent-antibody assay (IFA) or conventional enzyme immunoassays (EIAs) and by independent identification of autoantibodies in well-characterized samples. To achieve this goal, 984 samples were tested for seven analytes (SS/A, SS/B, Sm, RNP, Scl-70, double-stranded DNA [dsDNA], and centromere B) in both traditional and bead-based assays. The average concordance for the different analytes was 91%, ranging from 81% (dsDNA) to 97% (centromere B). The average relative specificity and sensitivity for the analytes were also high, 92% and 81%, respectively. An examination of 93 “normal controls” demonstrated a 7% false-positive rate, which was comparable to IFA. Percentages of different autoantibodies found in patients with a variety of disease conditions (34 with calcinosis, Raynaud's phenomenon, esophageal dysmotility, sclerodactyly, and telangiectasia; 41 with mixed connective tissue disease; 24 with scleroderma; and 35 with Sjogren's syndrome) were well within the range expected from each group. A scrutiny of results from AtheNA and EIA and Farr results for 185 systemic lupus erythematosus samples revealed comparable results by both methods, with the exception of SS/A and dsDNA, where AtheNA had a higher percentage of SS/A-positive results compared to EIA (51% versus 29%) and a lower percentage of dsDNA-positive results (18% versus 28% at a cutoff of 5 IU/ml).
Phagocytosis in the common grazing soil amoeba Acanthamoeba castellanii was characterized by flow cytometry. Uptake of fluorescently labelled latex microbeads by cells was quantified by appropriate setting of thresholds on light scatter channels and, subsequently, on fluorescence histograms. Confocal laser scanning microscopy was used to verify the effectiveness of sodium azide as a control for distinguishing between cell surface binding and internalization of beads. It was found that binding of beads at the cell surface was complete within 5 min and 80% of cells had beads associated with them after 10 min. However, the total number of phagocytosed beads continued to rise up to 2 h. The prolonged increase in numbers of beads phagocytosed was due to cell populations containing increasing numbers of beads peaking at increasing time intervals from the onset of phagocytosis. Fine adjustment of thresholds on light scatter channels was used to fractionate cells according to cell volume (cell cycle stage). Phagocytotic activity was approximately threefold higher in the largest (oldest) than in the smallest (newly divided) cells of A. castellanii and showed some evidence of periodicity. At no stage in the cell cycle did phagocytosis cease. Binding and phagocytosis of beads were also markedly influenced by culture age and rate of rotary agitation of cell suspensions. Saturation of phagocytosis (per cell) at increasing bead or decreasing cell concentrations occurred at bead/cell ratios exceeding 10:1. This was probably a result of a limitation of the vacuolar uptake system of A. castellanii, as no saturation of bead binding was evident. The advantages of flow cytometry for characterization of phagocytosis at the single-cell level in heterogeneous protozoal populations and the significance of the present results are discussed.
In this paper with the aid of negative dielectrophoresis force in conjuction with shear force and at an optimal sodium hydroxide concentration we demonstrated a switch-like functionality to elute specifically-bound beads from the surface. At an optimal flow rate and sodium hydroxide concentration, negative dielectrophoresis turned on results in bead detachment, whereas when negative dielectrophoresis is off, the beads remain attached. This platform offers the potential for performing a bead-based multiplexed assay where in a single channel various regions are immobilized with a different antibody, each targeting a different antigen. To develop the proof of concept and to demonstrate the switch-like functionality in eluting specifically-bound beads from the surface we looked at two different protein interactions. We chose interactions that were in the same order of magnitude in strength as typical antibody-antigen interactions. The first was Protein G-IgG interaction, and the second was the interaction between anti-IgG and IgG.
Binding of hepatitis C virus (HCV) RNA to core, the capsid protein, results in the formation of the nucleocapsid, the first step in the assembly of the viral particle. A novel assay was developed to discover small molecule inhibitors of core dimerization. This assay is based on time-resolved fluorescence resonance energy transfer (TR-FRET) between anti-tag antibodies labeled with either europium cryptate (Eu) or allophycocyanin (XL-665). The N-terminal 106-residue portion of core protein (core106) was tagged with either glutathione-S-transferase (GST) or a Flag peptide. Tag-free core106 was selected as the reference inhibitor. The assay was used to screen the library of pharmacologically active compounds (LOPAC) consisting of 1,280 compounds and a 2,240-compound library from the Center for Chemical Methodology and Library Development at Boston University (CMLD-BU). Ten of the 28 hits from the primary TR-FRET run were confirmed in a secondary amplified luminescent proximity homogeneous assay (ALPHA screen). One hit was further characterized by dose–response analysis yielding an IC50 of 9.3 μM. This 513 Da compound was shown to inhibit HCV production in cultured hepatoma cells.