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Journal articles on the topic 'Assay platform'

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Published: 4 June 2021
Last updated: 15 February 2022

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1

Chaby, Lauren E., Heather C. Lasseter, Kévin Contrepois, Reza M. Salek, Christoph W. Turck, Andrew Thompson, Timothy Vaughan, Magali Haas, and Andreas Jeromin. "Cross-Platform Evaluation of Commercially Targeted and Untargeted Metabolomics Approaches to Optimize the Investigation of Psychiatric Disease." Metabolites 11, no. 9 (September 8, 2021): 609. http://dx.doi.org/10.3390/metabo11090609.

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Metabolomics methods often encounter trade-offs between quantification accuracy and coverage, with truly comprehensive coverage only attainable through a multitude of complementary assays. Due to the lack of standardization and the variety of metabolomics assays, it is difficult to integrate datasets across studies or assays. To inform metabolomics platform selection, with a focus on posttraumatic stress disorder (PTSD), we review platform use and sample sizes in psychiatric metabolomics studies and then evaluate five prominent metabolomics platforms for coverage and performance, including intra-/inter-assay precision, accuracy, and linearity. We found performance was variable between metabolite classes, but comparable across targeted and untargeted approaches. Within all platforms, precision and accuracy were highly variable across classes, ranging from 0.9–63.2% (coefficient of variation) and 0.6–99.1% for accuracy to reference plasma. Several classes had high inter-assay variance, potentially impeding dissociation of a biological signal, including glycerophospholipids, organooxygen compounds, and fatty acids. Coverage was platform-specific and ranged from 16–70% of PTSD-associated metabolites. Non-overlapping coverage is challenging; however, benefits of applying multiple metabolomics technologies must be weighed against cost, biospecimen availability, platform-specific normative levels, and challenges in merging datasets. Our findings and open-access cross-platform dataset can inform platform selection and dataset integration based on platform-specific coverage breadth/overlap and metabolite-specific performance.
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2

Bosse, Roger, Russell Garlick, Beverly Brown, and Luc Menard. "Development of Nonseparation Binding and Functional Assays for G Protein-Coupled Receptors for High Throughput Screening: Pharmacological Characterization of the Immobilized CCR5 Receptor on FlashPlate(r)." Journal of Biomolecular Screening 3, no. 4 (June 1998): 285–92. http://dx.doi.org/10.1177/108705719800300407.

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G protein-coupled receptors (GPCRs) represent a very important class of drug targets. The development of microformatted nonseparation assays constitute a key step in the process of assay development for high throughput drug screening (HTS). We have developed a microformatted nonseparation assay for membrane preparations containing the CCR5 GPCR using FlashPlate® microplates (Packard Instrument Company, Meriden, CT). The pharmacodynamic (radioligand-binding) and functional (agonist-stimulated [35S]GTPγS binding) properties of this receptor observed in FlashPlate-based assays were compared with standard filtration assays. Saturation binding experiments performed using either assay platform revealed identical Kd for [125I]-MIP-1 β (0.7 nM). Comparable signal-to-noise ratios (SNR), similar affinities (Ki), and identical order of potency (RANTES ≅ MIP-1β > MIP-1α) were observed following competition binding assays in both platforms. In functional assays, the order of potency for different agonists were similar in both platforms with RANTES ≅ MIP-1β ≥ MIP-1α, which correspond to the relative affinities determined for the three ligands in competition binding experiments. Because similar pharmacologic properties were obtained in both FlashPlate microplates and standard filtration platforms, we conclude that FlashPlate microplates could provide a valuable nonseparation platform for primary and secondary HTS for this and possibly other GPCRs.
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Collet-Brose, Justine, Pierre-Jean Couble, Maureen R. Deehan, Robert J. Nelson, Walter G. Ferlin, and Sabrina Lory. "Evaluation of Multiple Immunoassay Technology Platforms to Select the Anti-Drug Antibody Assay Exhibiting the Most Appropriate Drug and Target Tolerance." Journal of Immunology Research 2016 (2016): 1–15. http://dx.doi.org/10.1155/2016/5069678.

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The aim of this study was, at the assay development stage and thus with an appropriate degree of rigor, to select the most appropriate technology platform and sample pretreatment procedure for a clinical ADA assay. Thus, ELISA, MSD, Gyrolab, and AlphaLISA immunoassay platforms were evaluated in association with target depletion and acid dissociation sample pretreatment steps. An acid dissociation step successfully improved the drug tolerance for all 4 technology platforms and the required drug tolerance was achieved with the Gyrolab and MSD platforms. The target tolerance was shown to be better for the ELISA format, where an acid dissociation treatment step alone was sufficient to achieve the desired target tolerance. However, inclusion of a target depletion step in conjunction with the acid treatment raised the target tolerance to the desired level for all of the technologies. A higher sensitivity was observed for the MSD and Gyrolab assays and the ELISA, MSD, and Gyrolab all displayed acceptable interdonor variability. This study highlights the usefulness of evaluating the performance of different assay platforms at an early stage in the assay development process to aid in the selection of the best fit-for-purpose technology platform and sample pretreatment steps.
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Dunn, D., M. Orlowski, P. McCoy, F. Gastgeb, K. Appell, L. Ozgur, M. Webb, and J. Burbaum. "Ultra-High Throughput Screen of Two-Million-Member Combinatorial Compound Collection in a Miniaturized, 1536-Well Assay Format." Journal of Biomolecular Screening 5, no. 3 (June 2000): 177–87. http://dx.doi.org/10.1177/108705710000500310.

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Results of a complete survey of the more than 2-million-member Pharmacopeia compound collection in a 1536-well microvolume screening assay format are reported. A complete technology platform, enabling the performance of ultra-high throughput screening in a miniaturized 1536-well assay format, has been assembled and utilized. The platform consists of tools for performing microvolume assays, including assay plates, liquid handlers, optical imagers, and data management software. A fluorogenic screening assay for inhibition of a protease enzyme target was designed and developed using this platform. The assay was used to perform a survey screen of the Pharmacopeia compound collection for active inhibitors of the target enzyme. The results from the survey demonstrate the successful implementation of the ultra-high throughout platform for routine screening purposes. Performance of the assay in the miniaturized format is equivalent to that of a standard 96-well assay, showing the same dependence on kinetic parameters and ability to measure enzyme inhibition. The survey screen identified an active class of compounds within the Pharmacopeia compound collection. These results were confirmed using a standard 96-well assay.
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Thompson, Iain, John McGiven, Jason Sawyer, Rachel Thirlwall, Nicola Commander, and Judy Stack. "Competitive Electrochemiluminescence Wash and No-Wash Immunoassays for Detection of Serum Antibodies to Smooth Brucella Strains." Clinical and Vaccine Immunology 16, no. 5 (March 4, 2009): 765–71. http://dx.doi.org/10.1128/cvi.00006-09.

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ABSTRACT Brucellosis is a bacterial zoonotic disease of major global importance. Natural hosts for Brucella species include animals of economic significance, such as cattle and small ruminants. Controlling brucellosis in natural hosts by high-throughput serological testing followed by the slaughter of seropositive animals helps to prevent disease transmission. This study aimed to convert an existing competitive enzyme-linked immunosorbent assay (cELISA), used for the serodiagnosis of brucellosis in ruminants, to two electrochemiluminescence (ECL) immunoassays on the Meso Scale Discovery (MSD) platform. The first assay employed a conventional plate washing step as part of the protocol. The second was a no-wash assay, made possible by the proximity-based nature of ECL signal generation by the MSD platform. Both ECL wash and no-wash assays closely matched the parent cELISA for diagnostic sensitivity and specificity. The results also demonstrated that both ECL assays met World Organization for Animal Health (OIE) standards, as defined by results for the OIE standard serum (OIEELISASPSS). This report is the first to describe an ECL assay incorporating lipopolysaccharide, an ECL assay for serodiagnosis of a bacterial infectious disease, a separation-free (no-wash) ECL assay for the detection of serum antibodies, and the use of the MSD platform for serodiagnosis. The simple conversion of the cELISA to the MSD platform suggests that many other serodiagnostic tests could readily be converted. Furthermore, the alignment of these results with the multiplex capability of the MSD platform offers the potential of no-wash multiplex assays to screen for several diseases.
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6

Zhao, Yanan, Yoji Nagasaki, Milena Kordalewska, Ellen G. Press, Ryan K. Shields, M. Hong Nguyen, Cornelius J. Clancy, and David S. Perlin. "Rapid Detection ofFKS-Associated Echinocandin Resistance in Candida glabrata." Antimicrobial Agents and Chemotherapy 60, no. 11 (August 22, 2016): 6573–77. http://dx.doi.org/10.1128/aac.01574-16.

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ABSTRACTA novel and highly accurate diagnostic assay platform was established for rapid identification ofFKSmutations associated with echinocandin resistance inCandida glabrata. The assay platform uses allele-specific molecular beacon and DNA melt analysis following asymmetric PCR. A dual assay forFKS1andFKS2was developed to identify within 3 h the most common and clinically relevant resistance-associated mutations, including 8FKS1HS1 (wild type [WT], S629P, F625S, D632Y, D632E [T1896G], D632E [T1896A], I634V, and F625F) and 7FKS2HS1 (WT, F659del, F659S, F659V, F659L, S663P, and S663F) genotypes. A blinded panel of 188C. glabrataclinical isolates was tested by both assays. The molecular diagnostic results from the dual assay were 100% concordant with data obtained from DNA sequencing. This platform has the potential to overcome the deficiencies of existingin vitrosusceptibility-based assays to identify echinocandin-resistantC. glabrataand holds promise as a surrogate diagnostic method to better direct echinocandin therapy.
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Ward, Matthew D., Kristin E. Mullins, Elizabeth Pickett, VeRonika Merrill, Mark Ruiz, Heather Rebuck, Show-Hong Duh, and Robert H. Christenson. "Performance of 4 Automated SARS-CoV-2 Serology Assay Platforms in a Large Cohort Including Susceptible COVID-19–Negative and COVID-19–Positive Patients." Journal of Applied Laboratory Medicine 6, no. 4 (March 10, 2021): 942–52. http://dx.doi.org/10.1093/jalm/jfab014.

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Abstract Background Anti–SARS-CoV-2 serological responses may have a vital role in controlling the spread of the disease. However, the comparative performance of automated serological assays has not been determined in susceptible patients with significant comorbidities. Methods In this study, we used large numbers of samples from patients who were negative (n = 2030) or positive (n = 112) for COVID-19 to compare the performance of 4 serological assay platforms: Siemens Healthineers Atellica IM Analyzer, Siemens Healthineers Dimension EXL Systems, Abbott ARCHITECT, and Roche cobas. Results All 4 serology assay platforms exhibited comparable negative percentage of agreement with negative COVID-19 status ranging from 99.2% to 99.7% and positive percentage of agreement from 84.8% to 87.5% with positive real-time reverse transcriptase PCR results. Of the 2142 total samples, only 38 samples (1.8%) yielded discordant results on one or more platforms. However, only 1.1% (23/2030) of results from the COVID-19–negative cohort were discordant. whereas discordance was 10-fold higher for the COVID-19–positive cohort, at 11.3% (15/112). Of the total 38 discordant results, 34 were discordant on only one platform. Conclusions Serology assay performance was comparable across the 4 platforms assessed in a large population of patients who were COVID-19 negative with relevant comorbidities. The pattern of discordance shows that samples were discordant on a single assay platform, and the discordance rate was 10-fold higher in the population that was COVID-19 positive.
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Suhandynata, Raymond T., Melissa A. Hoffman, Michael J. Kelner, Ronald W. McLawhon, Sharon L. Reed, and Robert L. Fitzgerald. "Multi-Platform Comparison of SARS-CoV-2 Serology Assays for the Detection of COVID-19." Journal of Applied Laboratory Medicine 5, no. 6 (August 7, 2020): 1324–36. http://dx.doi.org/10.1093/jalm/jfaa139.

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Abstract Background COVID-19 is caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), a novel beta-coronavirus that is responsible for the 2019 coronavirus pandemic. Acute infections should be diagnosed by polymerase chain reaction (PCR) based tests, but serology tests can demonstrate previous exposure to the virus. Methods We compared the performance of the Diazyme, Roche, and Abbott SARS-CoV-2 serology assays using 179 negative participants to determine negative percentage agreement (NPA) and in 60 SARS-CoV-2 PCR-confirmed positive patients to determine positive percentage agreement (PPA) at 3 different time frames following a positive SARS-CoV-2 PCR result. Results At ≥15 days, the PPA (95% CI) was 100 (86.3–100)% for the Diazyme IgM/IgG panel, 96.0 (79.7–99.9)% for the Roche total Ig assay, and 100 (86.3–100)% for the Abbott IgG assay. The NPA (95% CI) was 98.3 (95.2–99.7)% for the Diazyme IgM/IgG panel, 99.4 (96.9–100)% for the Roche total Ig assay, and 98.9 (96.0–99.9)% for the Abbott IgG assay. When the Roche total Ig assay was combined with either the Diazyme IgM/IgG panel or the Abbott IgG assay, the positive predictive value was 100% while the negative predictive value remained greater than 99%. Conclusions Our data demonstrates that the Diazyme, Roche, and Abbott SARS-CoV-2 serology assays have similar clinical performances. We demonstrated a low false-positive rate across all 3 platforms and observed that false positives observed on the Roche platform are unique compared to those observed on the Diazyme or Abbott assays. Using multiple platforms in tandem increases the PPVs, which is important when screening populations with low disease prevalence.
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Hemmila, Ilkka. "LANCE™: Homogeneous Assay Platform for HTS." Journal of Biomolecular Screening 4, no. 6 (December 1999): 303–7. http://dx.doi.org/10.1177/108705719900400604.

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10

Chen, Fang-yuan, Zheng Wang, Peng Li, Hong-Zhen Lian, and Hong-Yuan Chen. "Aptamer-based thrombin assay on microfluidic platform." ELECTROPHORESIS 34, no. 24 (November 18, 2013): 3260–66. http://dx.doi.org/10.1002/elps.201300338.

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Zhang, Bao Yue, Hui Xue Song, Tan Chen, and Zhan Hui Wang. "A Microfluidic Platform for Multi-Antigen Immunofluorescence Assays." Applied Mechanics and Materials 108 (October 2011): 200–205. http://dx.doi.org/10.4028/www.scientific.net/amm.108.200.

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Traditional cell-based assays such as cell immunoassay that utilizes plastic (chamber slides, dishes, microtiter plates), Magnetic bead, enzyme-linked immunsorbent assays (ELISA) [1], FACS cell sorting is labor intensive, time consuming, and requires a large numbers of cells or reagents. In this report, a microfluidic device integrated with cell culture, washing, fixation, and antigen-antibody reaction is presented for high-throughput immunoassay. Using this microfluidic device, each assay can be performed on a small number of cells and nanolitre or picolitre of reagents, this is particularly beneficial for rare or expensive cell types such as stem cells, or flow sorted cell populations. The capability of the microfluidic device was demonstrated for seeding human umbilical cord blood mesenchymal stem cells (UC-MSCs) in chambers and detecting the expression of surface markers (CD34, CD44, CD45, CD73, CD105, HLA-DR) by immunofluorescence assay.
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Veloria, John, Minhye Shin, Ashwini K. Devkota, Shelley M. Payne, Eun Jeong Cho, and Kevin N. Dalby. "Developing Colorimetric and Luminescence-Based High-Throughput Screening Platforms for Monitoring the GTPase Activity of Ferrous Iron Transport Protein B (FeoB)." SLAS DISCOVERY: Advancing the Science of Drug Discovery 24, no. 5 (April 30, 2019): 597–605. http://dx.doi.org/10.1177/2472555219844572.

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Iron is an essential requirement for the survival and virulence for bacteria. The bacterial ferrous iron transporter protein B (FeoB) functions as a major iron transporter in prokaryotes and has an N-terminal domain (NFeoB) with homology to eukaryotic G-proteins. Its GTPase activity is required for ferrous iron uptake, making it a potential target for antivirulence therapies. Here, two assay strategies relying on different spectroscopic readouts are described to monitor NFeoB GTPase activity. The first one is the colorimetric-based platform that utilizes a malachite green reagent to monitor phosphate production from GTP hydrolysis. The absorbance change directly relates to the GTPase activity of NFeoB. The assay was further improved by the addition of Tween-20 and miniaturized in a 384-well plate format with a 10 µL assay volume. The second format is a luminescence-based platform, measuring the GTP depletion by using a modified GTPase-Glo assay from Promega. In this platform, the luminescence signal correlates to the amount of GTP remaining, allowing for the direct calculation of GTP hydrolysis by NFeoB. The colorimetric platform was tested in a high-throughput manner against a custom-assembled library of a~2000 small molecules and was found to be simple, cost-effective, and robust. Additionally, the luminescence-based platform demonstrated its capability as an orthogonal assay to monitor GTPase activity, providing a valid and convenient method to filter false hits. These two assay platforms are proven to offset the limitations of each platform while enhancing overall quality and success rates.
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Satterly, Neal G., Matthew A. Voorhees, Abbe D. Ames, and Randal J. Schoepp. "Comparison of MagPix Assays and Enzyme-Linked Immunosorbent Assay for Detection of Hemorrhagic Fever Viruses." Journal of Clinical Microbiology 55, no. 1 (October 19, 2016): 68–78. http://dx.doi.org/10.1128/jcm.01693-16.

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ABSTRACT Viral hemorrhagic fevers, because of their high mortality rates, the lack of medical countermeasures, and their potential use as instruments of bioterrorism, pose a significant threat to the developed and the developing areas of the world. The key to preventing the spread of these diseases is early and accurate detection. For decades, the gold-standard immunoassay for hemorrhagic fever detection has been the enzyme-linked immunosorbent assay (ELISA); however, newer technologies are emerging with increased sensitivities. One such technology is the Luminex MagPix platform using xMAP microspheres. Here, we compare the MagPix platform with a traditional ELISA for IgM and antigen detection of infections from Lassa and Ebola viruses (LASV and EBOV, respectively). For IgM detection in nonhuman primate samples, the MagPix platform was 5 and 25 times more sensitive in detecting LASV and EBOV, respectively, compared to that with ELISA. For antigen detection in buffer, the MagPix platform was 25 and 2.5 times more sensitive in detecting lower levels of LASV and EBOV, respectively. In both IgM and antigen detection assays, the MagPix platform demonstrated excellent reproducibility at the lower limit of detection (LLOD). These findings demonstrate that the MagPix platform is a viable diagnostic replacement for the ELISA for viral hemorrhagic fevers.
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Boehnke, Karsten, Philip W. Iversen, Dirk Schumacher, María José Lallena, Rubén Haro, Joaquín Amat, Johannes Haybaeck, et al. "Assay Establishment and Validation of a High-Throughput Screening Platform for Three-Dimensional Patient-Derived Colon Cancer Organoid Cultures." Journal of Biomolecular Screening 21, no. 9 (July 10, 2016): 931–41. http://dx.doi.org/10.1177/1087057116650965.

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The application of patient-derived three-dimensional culture systems as disease-specific drug sensitivity models has enormous potential to connect compound screening and clinical trials. However, the implementation of complex cell-based assay systems in drug discovery requires reliable and robust screening platforms. Here we describe the establishment of an automated platform in 384-well format for three-dimensional organoid cultures derived from colon cancer patients. Single cells were embedded in an extracellular matrix by an automated workflow and subsequently self-organized into organoid structures within 4 days of culture before being exposed to compound treatment. We performed validation of assay robustness and reproducibility via plate uniformity and replicate-experiment studies. After assay optimization, the patient-derived organoid platform passed all relevant validation criteria. In addition, we introduced a streamlined plate uniformity study to evaluate patient-derived colon cancer samples from different donors. Our results demonstrate the feasibility of using patient-derived tumor samples for high-throughput assays and their integration as disease-specific models in drug discovery.
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Zhang, Hongkang, Bryan D. Moyer, Violeta Yu, Joseph G. McGivern, Michael Jarosh, Christopher A. Werley, Vivian C. Hecht, et al. "Correlation of Optical and Automated Patch Clamp Electrophysiology for Identification of NaV1.7 Inhibitors." SLAS DISCOVERY: Advancing the Science of Drug Discovery 25, no. 5 (April 15, 2020): 434–46. http://dx.doi.org/10.1177/2472555220914532.

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The voltage-gated sodium channel Nav1.7 is a genetically validated target for pain; pharmacological blockers are promising as a new class of nonaddictive therapeutics. The search for Nav1.7 subtype selective inhibitors requires a reliable, scalable, and sensitive assay. Previously, we developed an all-optical electrophysiology (Optopatch) Spiking HEK platform to study activity-dependent modulation of Nav1.7 in a format compatible with high-throughput screening. In this study, we benchmarked the Optopatch Spiking HEK assay with an existing validated automated electrophysiology assay on the IonWorks Barracuda (IWB) platform. In a pilot screen of 3520 compounds, which included compound plates from a random library as well as compound plates enriched for Nav1.7 inhibitors, the Optopatch Spiking HEK assay identified 174 hits, of which 143 were confirmed by IWB. The Optopatch Spiking HEK assay maintained the high reliability afforded by traditional fluorescent assays and further demonstrated comparable sensitivity to IWB measurements. We speculate that the Optopatch assay could provide an affordable high-throughput screening platform to identify novel Nav1.7 subtype selective inhibitors with diverse mechanisms of action, if coupled with a multiwell parallel optogenetic recording instrument.
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Tan, Charles Y., Fred W. Immermann, Shite Sebastian, Michael W. Pride, Danka Pavliakova, Kelly A. Belanger, Wendy Watson, et al. "Evaluation of a Validated Luminex-Based Multiplex Immunoassay for Measuring Immunoglobulin G Antibodies in Serum to Pneumococcal Capsular Polysaccharides." mSphere 3, no. 4 (August 8, 2018): e00127-18. http://dx.doi.org/10.1128/msphere.00127-18.

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ABSTRACT This article describes the results of a study designed to bridge the World Health Organization (WHO) pneumococcal enzyme-linked immunosorbent assay (ELISA) platform to the validated Luminex-based 13-plex direct immunoassay (dLIA) platform developed by Pfizer, Inc. Both assay platforms quantify serotype-specific serum IgG antibodies (in micrograms per milliliter) against an international reference standard serum. The primary goal of this study was to determine if the dLIA is a suitable replacement for the ELISA to support clinical vaccine studies that include the evaluation of immune responses to serotypes 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, and 23F. Serum samples were selected from four pivotal 13-valent pneumococcal conjugate vaccine (13vPnC; Prevnar 13) clinical trials on the basis of their serotype-specific IgG concentrations by ELISA. In these studies, subjects were immunized either with 13vPnC or with 7-valent pneumococcal conjugate vaccine (7vPnC; Prevnar). There were 1,528 of 1,574 selected samples with sufficient remaining volume for reanalysis in the dLIA. A comparison of assay results from the dLIA and ELISA platforms showed clear and robust linear quantitative relationships across all 13 serotypes. In addition, lower IgG antibody concentrations in preimmunization samples were measured in the dLIA, thus allowing better differentiation between preimmunization and low-titer postimmunization samples. Overall, the results showed that the established population-level protective threshold IgG concentration, 0.35 µg/ml of serotype-specific serum IgG antibodies, is appropriate for use for data generated using the dLIA platform developed by Pfizer, Inc., for 10 serotypes: serotypes 1, 3, 4, 6A, 7F, 9V, 14, 18C, 19F, and 23F. On the basis of the extensive bridging analyses, however, the use of dLIA cutoff values of 0.23, 0.10, and 0.12 µg/ml is recommended for serotypes 5, 6B, and 19A, respectively. This adjustment will ensure that the consistency of the established population-level protective threshold IgG concentration is maintained when switching from the ELISA to the dLIA platform. The results of this bridging study demonstrate that the 13-plex dLIA platform is a suitable replacement for the WHO reference ELISA platform. IMPORTANCE The pneumococcal enzyme-linked immunosorbent assay (ELISA) measures IgG antibodies in human serum, and it is an important assay that supports licensure of pneumococcal vaccines. The immune correlate of protection, 0.35 µg/ml of IgG antibodies, was determined by the ELISA method. Pfizer has developed a new Luminex-based assay platform to replace the ELISA. These papers describe the important work of (i) validating the Luminex-based assay and (ii) bridging the immune correlate of protection (0.35 µg/ml IgG) to equivalent values reported by the Luminex platform.
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Lee, Sang-Yun, Il Doh, and Dong Woo Lee. "A High Throughput Apoptosis Assay using 3D Cultured Cells." Molecules 24, no. 18 (September 16, 2019): 3362. http://dx.doi.org/10.3390/molecules24183362.

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A high throughput apoptosis assay using 3D cultured cells was developed with a micropillar/microwell chip platform. Live cell apoptosis assays based on fluorescence detection have been useful in high content screening. To check the autofluorescence of drugs, controls (no caspase-3/7 reagent in the assay) for the drugs are necessary which require twice the test space. Thus, a high throughput capability and highly miniaturized format for reducing reagent usage are necessary in live cell apoptosis assays. Especially, the expensive caspase-3/7 reagent should be reduced in a high throughput screening system. To solve this issue, we developed a miniaturized apoptosis assay using micropillar/microwell chips for which we tested seventy drugs (six replicates) per chip and reduced the assay volume to 1 µL. This reduced assay volume can decrease the assay costs compared to the 10–40 µL assay volumes used in 384 well plates. In our experiments, among the seventy drugs, four drugs (Cediranib, Cabozatinib, Panobinostat, and Carfilzomib) induced cell death by apoptosis. Those results were confirmed with western blot assays and proved that the chip platform could be used to identify high potency apoptosis-inducing drugs in 3D cultured cells with alginate.
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Tallmadge, Rebecca L., Renee Anderson, Patrick K. Mitchell, Zachary C. Forbes, Brenda Werner, Gloria Gioia, Paolo Moroni, Amy Glaser, Anil J. Thachil, and Laura B. Goodman. "Characterization of a novel Mycoplasma cynos real-time PCR assay." Journal of Veterinary Diagnostic Investigation 32, no. 6 (November 21, 2019): 793–801. http://dx.doi.org/10.1177/1040638719890858.

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Mycoplasma cynos is recognized as an emerging causative pathogen of canine infectious respiratory disease (CIRD) worldwide. We developed a new open-source real-time PCR (rtPCR) assay for M. cynos that performs well under standard rtPCR conditions. Primers and probes were designed to target the M. cynos tuf gene. Reaction efficiencies for the M. cynos tuf gene assay on 2 platforms were based on amplification of standard curves spanning 8 orders of magnitude: ABI 7500 platform, 94.3–97.9% ( r2 ≥ 0.9935); QuantStudio OpenArray platform, 119.1–122.5% ( r2 = 0.9784). The assay performed very well over a range of template input, from 109 copies to the lower limit of quantification at 4 copies of the M. cynos genome on the ABI 7500 platform. Diagnostic performance was estimated by comparison with an in-house legacy assay on clinical specimens as well as testing isolates that were characterized previously by intergenic spacer region (ISR) sequencing. Exclusivity was established by testing 12 other Mycoplasma species. To substantiate the high specificity of the M. cynos tuf gene assay, sequence confirmation was performed on ISR PCR amplicons obtained from clinical specimens. One ISR amplicon sequence revealed M. mucosicanis rather than M. cynos. The complete protocol of the newly developed M. cynos tuf assay is provided to facilitate assay harmonization.
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Jensen, Steffen Grann, Samantha Epistolio, Cesilie Lind Madsen, Majbritt Hauge Kyneb, Alice Riva, Alessia Paganotti, Jessica Barizzi, et al. "A new sensitive and fast assay for the detection of EGFR mutations in liquid biopsies." PLOS ONE 16, no. 6 (June 24, 2021): e0253687. http://dx.doi.org/10.1371/journal.pone.0253687.

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Background A major perspective for the use of circulating tumor DNA (ctDNA) in the clinical setting of non-small cell lung cancer (NSCLC) is expected as predictive factor for resistance and response to EGFR TKI therapy and, especially, as a non-invasive alternative to tissue biopsy. However, ctDNA is both highly fragmented and mostly low concentrated in plasma and serum. On this basis, it is important to use a platform characterized by high sensitivity and linear performance in the low concentration range. This motivated us to evaluate the newly developed and commercially available SensiScreen® EGFR Liquid assay platform (PentaBase) with regard to sensitivity, linearity, repeatability and accuracy and finally to compare it to our already implemented methods. The validation was made in three independent European laboratories using two cohorts on a total of 68 unique liquid biopsies. Results Using artificial samples containing 1600 copies of WT DNA spiked with 50% - 0.1% of mutant copies across a seven—log dilution scale, we assessed the sensitivity, linearity, repeatability and accuracy for the p.T790M, p.L858R and exon 19 deletion assays of the SensiScreen® EGFR Liquid assay platform. The lowest value detectable ranged from 0.5% to 0.1% with R2≥0,97 indicating good linearity. High PCR efficiency was shown for all three assays. In 102 single PCRs each containing theoretical one copy of the mutant at initiating, assays showed repeatable positivity in 75.5% - 80.4% of reactions. At low ctDNA levels, as in plasma, the SensiScreen® EGFR Liquid assay platform showed better sensitivity than the Therascreen® EGFR platform (Qiagen) and equal performance to the ctEGFR Mutation Detection Kit (EntroGen) and the IOT® Oncomine cell-free nucleic acids assay (Thermo Fisher Scientific) with 100% concordance at the sequence level. Conclusion For profiling clinical plasma samples, characterized by low ctDNA abundance, the SensiScreen® EGFR Liquid assay is able to identify down to 1 copy of mutant alleles and with its high sensitivity, linearity and accuracy it may be a competitive platform of choice.
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Shapiro, M. S., X. Wang, D. R. Mendu, and A. Firpo. "Multiplex Assays of Luminex for Identification of COVID-19 Antibodies in Patient Serum: Performance Evaluation and Comparison to ELISA." American Journal of Clinical Pathology 154, Supplement_1 (October 2020): S92. http://dx.doi.org/10.1093/ajcp/aqaa161.201.

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Abstract Introduction/Objective Mount Sinai Hospital has received emergency use authorization (EUA) from the FDA for Coronavirus Disease 2019 (COVID-19) antibody testing using ELISA. This serological assay detects and titrates the presence of circulating antibodies to COVID-19. Other platforms have aimed to achieve the credentials of the ELISA instrument, including the multiplex assays of Luminex. The platform is known to have a greater throughput (384 wells vs. 96 wells per microplate) and faster processing speed (8 hours vs. 17 hours). Methods Luminex utilizes beads that couple to the same COVID-19 antigens (mRBD and mSpike) which were utilized for the ELISA assay. The beads are read determining the mean fluorescence intensity (MFI). In order to compare the two methods, our study included 61 patients with COVID-19 at Mount Sinai Hospital, to screen and titrate their sera using Luminex, and to correspond the MFI values with the ELISA titers. Results The Luminex assay has achieved the same level of confidence as ELISA. The 61 patients, representing 30 negatives and 31 positives, are consistently identified as such on both platforms. Our data highlights 32% of patients with a low titer (<1:160), 42% of patients with a high titer (1:160 ~ 1:320), and 26% of patients with a very high titer level (>1:320). These titers correlated well with the MFI values. Based on a cutoff of 80,000 MFI, the sensitivity and specificity of the assay is 98% and 85%, respectively, with no overlapping of MFI between positive and negative results. Conclusion Overall, the study has demonstrated that the Luminex is a strong alternative for the ELISA platform. The Luminex highlights the broad dynamic range with no overlapping between positives and negatives. Migration from ELISA to Luminex, a platform with faster and greater throughput, is therefore, highly desirable.
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Pavliakova, Danka, Peter C. Giardina, Soraya Moghazeh, Shite Sebastian, Maya Koster, Viliam Pavliak, Andrew McKeen, Roger French, Kathrin U. Jansen, and Michael Pride. "Development and Validation of 13-plex Luminex-Based Assay for Measuring Human Serum Antibodies to Streptococcus pneumoniae Capsular Polysaccharides." mSphere 3, no. 4 (August 8, 2018): e00128-18. http://dx.doi.org/10.1128/msphere.00128-18.

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ABSTRACT A Luminex-based direct immunoassay (dLIA) platform has been developed to replace the standardized pneumococcal enzyme-linked immunosorbent assay platform. The multiplex dLIA simultaneously measures the concentration of serum immunoglobulin G (IgG) antibodies specific for pneumococcal capsular polysaccharide (PnPS) serotypes 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, and 23F. The assay uses poly-l-lysine (PLL)-conjugated PnPS, chemically coupled to spectrally distinct Luminex microspheres. Assay validation experiments were performed using residual human serum samples obtained from 13-valent pneumococcal conjugate vaccine (13vPnC) clinical studies. Assay results are expressed as IgG antibody concentrations in micrograms per milliliter using the international reference serum, 007sp. The lower limit of quantitation (LLOQ) for all serotypes covered in the 13-plex dLIA fell within the range of 0.002 to 0.038 µg/ml serum IgG. The difference between the lower limit and upper limit of the assay range was >500-fold for all serotypes, and assay variability was <20% relative standard deviation (RSD) for all serotypes. IgG antibody measurements were shown to be serotype-specific (some cross-reactivity was observed only between the structurally related serotypes 6A and 6B as well as 19A and 19F), and no interference was observed between the serotypes when the assay was performed in the 13-plex format compared to the singleplex assays. The 13-plex dLIA platform developed by Pfizer Inc. generates up to 143 test results in a single 96-well plate and is a suitable replacement of the enzyme-linked immunosorbent assay (ELISA) platform for evaluating vaccine clinical trials. IMPORTANCE The pneumococcal enzyme-linked immunosorbent assay (ELISA) measures IgG antibodies in human serum, and it is an important assay that supports licensure of pneumococcal vaccines. The immune correlate of protection, 0.35 µg/ml of IgG antibodies, was determined by the ELISA method. Pfizer has developed a new Luminex-based assay platform to replace the ELISA. These papers describe the important work of (i) validating the Luminex-based assay and (ii) bridging the immune correlate of protection (0.35 µg/ml IgG) to equivalent values reported by the Luminex platform.
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Janczak, Colleen M., Isen A. C. Calderon, Eka Noviana, Priyanka Hadvani, Joo Ryung Lee, and Craig A. Aspinwall. "Hybrid Nanoparticle Platform for Nanoscale Scintillation Proximity Assay." ACS Applied Nano Materials 2, no. 3 (February 20, 2019): 1259–66. http://dx.doi.org/10.1021/acsanm.8b02136.

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Du, Wen-Bin, Meng Sun, Shu-Qing Gu, Ying Zhu, and Qun Fang. "Automated Microfluidic Screening Assay Platform Based on DropLab." Analytical Chemistry 82, no. 23 (December 2010): 9941–47. http://dx.doi.org/10.1021/ac1020479.

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van Berkel, Sander S., Jürgen Brem, Anna M. Rydzik, Ramya Salimraj, Ricky Cain, Anil Verma, Raymond J. Owens, Colin W. G. Fishwick, James Spencer, and Christopher J. Schofield. "Assay Platform for Clinically Relevant Metallo-β-lactamases." Journal of Medicinal Chemistry 56, no. 17 (August 16, 2013): 6945–53. http://dx.doi.org/10.1021/jm400769b.

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Lambert-Messerlian, G. M., G. E. Palomaki, and J. A. Canick. "Inhibin A measurement using an automated assay platform." Prenatal Diagnosis 28, no. 5 (2008): 399–403. http://dx.doi.org/10.1002/pd.1983.

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Hu, Peng, Lei Han, Chengzhou Zhu, and Shao Jun Dong. "Nanoreactors: a novel biosensing platform for protein assay." Chemical Communications 49, no. 17 (2013): 1705. http://dx.doi.org/10.1039/c2cc37734a.

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Ganguly, Payel, Landiso Madonsela, Jesse T. Chao, Christopher J. R. Loewen, Timothy P. O’Connor, Esther M. Verheyen, and Douglas W. Allan. "A scalable Drosophila assay for clinical interpretation of human PTEN variants in suppression of PI3K/AKT induced cellular proliferation." PLOS Genetics 17, no. 9 (September 7, 2021): e1009774. http://dx.doi.org/10.1371/journal.pgen.1009774.

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Gene variant discovery is becoming routine, but it remains difficult to usefully interpret the functional consequence or disease relevance of most variants. To fill this interpretation gap, experimental assays of variant function are becoming common place. Yet, it remains challenging to make these assays reproducible, scalable to high numbers of variants, and capable of assessing defined gene-disease mechanism for clinical interpretation aligned to the ClinGen Sequence Variant Interpretation (SVI) Working Group guidelines for ‘well-established assays’. Drosophila melanogaster offers great potential as an assay platform, but was untested for high numbers of human variants adherent to these guidelines. Here, we wished to test the utility of Drosophila as a platform for scalable well-established assays. We took a genetic interaction approach to test the function of ~100 human PTEN variants in cancer-relevant suppression of PI3K/AKT signaling in cellular growth and proliferation. We validated the assay using biochemically characterized PTEN mutants as well as 23 total known pathogenic and benign PTEN variants, all of which the assay correctly assigned into predicted functional categories. Additionally, function calls for these variants correlated very well with our recent published data from a human cell line. Finally, using these pathogenic and benign variants to calibrate the assay, we could set readout thresholds for clinical interpretation of the pathogenicity of 70 other PTEN variants. Overall, we demonstrate that Drosophila offers a powerful assay platform for clinical variant interpretation, that can be used in conjunction with other well-established assays, to increase confidence in the accurate assessment of variant function and pathogenicity.
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Kuhn, P., SM Keating, GT Baxter, K. Thomas, A. Kolatkar, and CC Sigman. "Lessons Learned: Transfer of the High-Definition Circulating Tumor Cell Assay Platform to Development as a Commercialized Clinical Assay Platform." Clinical Pharmacology & Therapeutics 102, no. 5 (March 22, 2017): 777–85. http://dx.doi.org/10.1002/cpt.645.

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McCoy, Austin G., Timothy D. Miles, Guillaume J. Bilodeau, Patrick Woods, Cheryl Blomquist, Frank N. Martin, and Martin I. Chilvers. "Validation of a Preformulated, Field Deployable, Recombinase Polymerase Amplification Assay for Phytophthora Species." Plants 9, no. 4 (April 7, 2020): 466. http://dx.doi.org/10.3390/plants9040466.

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Recombinase polymerase amplification (RPA) assays are valuable molecular diagnostic tools that can detect and identify plant pathogens in the field without time-consuming DNA extractions. Historically, RPA assay reagents were commercially available as a lyophilized pellet in microfuge strip tubes, but have become available in liquid form more recently—both require the addition of primers and probes prior to use, which can be challenging to handle in a field setting. Lyophilization of primers and probes, along with RPA reagents, contained within a single tube limits the risk of contamination, eliminates the need for refrigeration, as the lyophilized reagents are stable at ambient temperatures, and simplifies field use of the assays. This study investigates the potential effect of preformulation on assay performance using a previously validated Phytophthora genus-specific RPA assay, lyophilized with primers and probes included with the RPA reagents. The preformulated lyophilized Phytophthora RPA assay was compared with a quantitative polymerase chain reaction (qPCR) assay and commercially available RPA kits using three qPCR platforms (BioRad CFX96, QuantStudio 6 and Applied Biosystems ViiA7) and one isothermal platform (Axxin T16-ISO RPA), with experiments run in four separate labs. The assay was tested for sensitivity (ranging from 500 to 0.33 pg of DNA) and specificity using purified oomycete DNA, as well as crude extracts of Phytophthora-infected and non-infected plants. The limit of detection (LOD) using purified DNA was 33 pg in the CFX96 and ViiA7 qPCR platforms using the preformulated kits, while the Axxin T16-ISO RPA chamber and the QuantStudio 6 platform could detect down to 3.3 pg with or without added plant extract. The LOD using a crude plant extract for the BioRad CFX96 was 330 pg, whereas the LOD for the ViiA7 system was 33 pg. These trials demonstrate the consistency and uniformity of pathogen detection with preformulated RPA kits for Phytophthora detection when conducted by different labs using different instruments for measuring results.
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Sanders, Karen A., Dimitar K. Gavrilov, Devin Oglesbee, Kimiyo M. Raymond, Silvia Tortorelli, John J. Hopwood, Fred Lorey, et al. "A Comparative Effectiveness Study of Newborn Screening Methods for Four Lysosomal Storage Disorders." International Journal of Neonatal Screening 6, no. 2 (May 30, 2020): 44. http://dx.doi.org/10.3390/ijns6020044.

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Newborn screening for one or more lysosomal disorders has been implemented in several US states, Japan and Taiwan by multiplexed enzyme assays using either tandem mass spectrometry or digital microfluidics. Another multiplex assay making use of immunocapture technology has also been proposed. To investigate the potential variability in performance of these analytical approaches, we implemented three high-throughput screening assays for the simultaneous screening for four lysosomal disorders: Fabry disease, Gaucher disease, mucopolysaccharidosis type I, and Pompe disease. These assays were tested in a prospective comparative effectiveness study using nearly 100,000 residual newborn dried blood spot specimens. In addition, 2nd tier enzyme assays and confirmatory molecular genetic testing were employed. Post-analytical interpretive tools were created using the software Collaborative Laboratory Integrated Reports (CLIR) to determine its ability to improve the performance of each assay vs. the traditional result interpretation based on analyte-specific reference ranges and cutoffs. This study showed that all three platforms have high sensitivity, and the application of CLIR tools markedly improves the performance of each platform while reducing the need for 2nd tier testing by 66% to 95%. Moreover, the addition of disease-specific biochemical 2nd tier tests ensures the lowest false positive rates and the highest positive predictive values for any platform.
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Schwers, Stephan, Elke Reifenberger, Mathias Gehrmann, Alexandre Izmailov, and Kerstin Bohmann. "A High-Sensitivity, Medium-Density, and Target Amplification–Free Planar Waveguide Microarray System for Gene Expression Analysis of Formalin-Fixed and Paraffin-Embedded Tissue." Clinical Chemistry 55, no. 11 (November 1, 2009): 1995–2003. http://dx.doi.org/10.1373/clinchem.2009.128215.

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Abstract Background: Many microarray platforms and their associated assay chemistries do not work properly with RNA extracted from formalin-fixed, paraffin-embedded (FFPE) tissue samples, a feature that severely hampers the use of microarrays in oncology applications, for which FFPE tissue is the routine specimen. Furthermore, the limited sensitivity of most microarray platforms requires time-consuming and costly amplification reactions of the target RNA, which negatively affects clinical laboratory work flow. Methods: We developed an approach for sensitively and reliably measuring mRNA abundances in FFPE tissue samples. This approach involves automated RNA extractions, direct hybridization of extracted RNA to immobilized capture probes, antibody-mediated labeling, and readout with an instrument applying the principle of planar waveguides (PWG). A 14-gene multiplex assay conducted with RNA isolated from 20 FFPE blocks was correlated to an analysis of the same with reverse-transcription quantitative real-time PCR (RT-qPCR). Results: The assay sensitivity for gene expression analysis obtained for the PWG microarray platform was &lt;10 fmol/L, eliminating the need for target preamplification. We observed a correlation coefficient of 0.87 to state-of-the-art RT-qPCR technology with RNA isolated from FFPE tissue, despite a compressed dynamic range for the PWG system (a 2.9-log dynamic range for PWG in our test system vs 5.0 logs for RT-qPCR). The precision of the PWG platform was comparable to RT-qPCR (Pearson correlation coefficient of 0.9851 for PWG vs 0.9896 for RT-qPCR) for technical replicates. Conclusions: The presented PWG platform demonstrated excellent sensitivity and precision and is especially well suited for any application for which fast, simple, and robust multiplex assays of RNA in FFPE tissue are required.
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Keiser, Patrick T., Manu Anantpadma, Hilary Staples, Ricardo Carrion, and Robert A. Davey. "Automation of Infectious Focus Assay for Determination of Filovirus Titers and Direct Comparison to Plaque and TCID50 Assays." Microorganisms 9, no. 1 (January 12, 2021): 156. http://dx.doi.org/10.3390/microorganisms9010156.

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Ongoing efforts to develop effective therapies against filoviruses rely, to different extents, on quantifying the amount of viable virus in samples by plaque, TCID50, and focus assays. Unfortunately, these techniques have inherent variance, and laboratory-specific preferences make direct comparison of data difficult. Additionally, human errors such as operator errors and subjective bias can further compound the differences in outcomes. To overcome these biases, we developed a computer-based automated image-processing method for a focus assay based on the open-source CellProfiler software platform, which enables high-throughput screening of many treatment samples at one time. We compared virus titers calculated using this platform to plaque and TCID50 assays using common stocks of virus for 3 major Filovirus species, Zaire ebolavirus, Sudan ebolavirus, and Marburg marburgvirus with each assay performed by multiple operators on multiple days. We show that plaque assays give comparable findings that differ by less than 3-fold. Focus-forming unit (FFU) and TCID50 assays differ by 10-fold or less from the plaque assays due a higher (FFU) and lower (TCID50) sensitivity. However, reproducibility and accuracy of each assay differs significantly with Neutral Red Agarose Overlay plaque assays and TCID50 with the lowest reproducibility due to subjective analysis and operator error. Both crystal violet methylcellulose overlay plaque assay and focus assays perform best for accuracy and the focus assay performs best for speed and throughput.
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Tang, Huaping, Reshma Panemangalore, Melissa Yarde, Litao Zhang, and Mary Ellen Cvijic. "384-Well Multiplexed Luminex Cytokine Assays for Lead Optimization." Journal of Biomolecular Screening 21, no. 6 (April 19, 2016): 548–55. http://dx.doi.org/10.1177/1087057116644164.

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Cytokines serve as a major mechanism of communication between immune cells and are the functional molecules at the end of immune pathways. Abnormalities in cytokines are involved in a wide variety of diseases, including chronic inflammation, autoimmune diseases, and cancer. Cytokines are not only direct targets of therapeutics but also important biomarkers for assessing drug efficacy and safety. Traditionally, enzyme-linked immunosorbent assays (ELISA) were most popular for identifying and quantifying cytokines. However, ELISA is expensive, labor intensive, and low throughput. Here, we report the development of a miniaturized Luminex (Austin, TX) assay platform to establish a panel of high-throughput, multiplexed assays for measuring cytokines in human whole blood. The miniaturized 384-well Luminex assay uses <25% of the assay reagents compared with the 96-well assay. The development and validation of the 384-well Luminex cytokine assays enabled high-throughput screening of compounds in primary cells using cytokines as physiologically relevant readouts. Furthermore, this miniaturized multiplexed technology platform allows for high-throughput biomarker profiling of biofluids from animal studies and patient samples for translational research.
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Rathore, Rakesh, Jay Corr, George Scott, Pauline Vollmerhaus, and Kenneth D. Greis. "Development of an Inhibitor Screening Platform via Mass Spectrometry." Journal of Biomolecular Screening 13, no. 10 (November 21, 2008): 1007–13. http://dx.doi.org/10.1177/1087057108326143.

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Commonly used methods for isolated enzyme inhibitor screening typically rely on fluorescent or chemiluminescent detection techniques that are often indirect and/or coupled assays. Mass spectrometry (MS) has been widely reported for measuring the conversion of substrates to products for enzyme assays and has more recently been demonstrated as an alternative readout system for inhibitor screening. In this report, a high-throughput mass spectrometry (HTMS) readout platform, based on the direct measurement of substrate conversion to product, is presented. The rapid ionization and desorption features of a new generation matrix-assisted laser desorption ionization-triple quadrupole (MALDI-QqQ) mass spectrometer are shown to improve the speed of analysis to greater than 1 sample per second while maintaining excellent Z′ values. Furthermore, the readout was validated by demonstrating the ability to measure IC50 values for several known kinase inhibitors against cyclic AMP—dependent protein kinase (PKA). Finally, when the assay performance was compared with a common ADPaccumulation readout system, this HTMS approach produced better signal-to-background ratios, higher Z′ values, and a reagent cost of about $0.03 per well compared with about $0.60 per well for the fluorescence assay. Collectively, these data demonstrate that a MALDI-QqQ-MS—based readout platform offers significant advantages over the commonly used assays in terms of speed, sensitivity, reproducibility, and reagent cost. ( Journal of Biomolecular Screening 2008:1007-1013)
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Osmond, Ronald I. W., Antony Sheehan, Romana Borowicz, Emma Barnett, Georgina Harvey, Cheryl Turner, Andrea Brown, Michael F. Crouch, and Anthony R. Dyer. "GPCR Screening via ERK 1/2: A Novel Platform for Screening G Protein–Coupled Receptors." Journal of Biomolecular Screening 10, no. 7 (September 16, 2005): 730–37. http://dx.doi.org/10.1177/1087057105277968.

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Discovery of novel agonists and antagonists for G protein–coupled receptors (GPCRs) relies heavily on cell-based assays because determination of functional consequences of receptor engagement is often desirable. Currently, there are several key parameters measured to achieve this, including mobilization of intracellular Ca2+ and formation of cyclic adenosine monophosphate or inositol triphosphate. However, no single assay platform is suitable for all situations, and all of the assays have limitations. The authors have developed a new high-throughput homogeneous assay platform for GPCR discovery as an alternative to current assays, which employs detection of phosphorylation of the key signaling molecule p42/44 MAP kinase (ERK 1/2). The authors show that ERK 1/2 is consistently activated in cells stimulated by Gq-coupled GPCRs and provides a new high-throughput platform for screening GPCR drug candidates. The activation of ERK 1/2 in Gq-coupled GPCR systems generates comparable pharmacological data for receptor agonist and antagonist data obtained by other GPCR activation measurement techniques.
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Lebakken, Connie S., Steven M. Riddle, Upinder Singh, W. Jack Frazee, Hildegard C. Eliason, Yi Gao, Laurie J. Reichling, Bryan D. Marks, and Kurt W. Vogel. "Development and Applications of a Broad-Coverage, TR-FRET-Based Kinase Binding Assay Platform." Journal of Biomolecular Screening 14, no. 8 (June 29, 2009): 924–35. http://dx.doi.org/10.1177/1087057109339207.

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The expansion of kinase assay technologies over the past decade has mirrored the growing interest in kinases as drug targets. As a result, there is no shortage of convenient, fluorescence-based methods available to assay targets that span the kinome. The authors recently reported on the development of a non-activity-based assay to characterize kinase inhibitors that depended on displacement of an Alexa Fluor® 647 conjugate of staurosporine (a “tracer”) from a particular kinase. Kinase inhibitors were characterized by a change in fluorescence lifetime of the tracer when it was bound to a kinase relative to when it was displaced by an inhibitor. Here, the authors report on improvements to this strategy by reconfiguring the assay in a time-resolved fluorescence resonance energy transfer (TR-FRET) format that simplifies instrumentation requirements and allows for the use of a substantially lower concentration of kinase than was required in the fluorescence-lifetime-based format. The authors use this new assay to demonstrate several aspects of the binding assay format that are advantageous relative to traditional activity-based assays. The TR-FRET binding format facilitates the assay of compounds against low-activity kinases, allows for the characterization of type II kinase inhibitors either using nonactivated kinases or by monitoring compound potency over time, and ensures that the signal being detected is specific to the kinase of interest and not a contaminating kinase.
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Jiang, Zhihua, John Kamerud, Zhiping You, Soma Basak, Elena Seletskaia, Gregory S. Steeno, and Boris Gorovits. "Feasibility of singlicate-based analysis in bridging ADA assay on Meso-Scale Discovery platform: comparison with duplicate analysis." Bioanalysis 13, no. 14 (July 2021): 1123–34. http://dx.doi.org/10.4155/bio-2021-0095.

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Aim: To investigate the feasibility of singlicate analysis in anti-drug antibody (ADA) assay by comparing performance characteristics for assays qualified in duplicate and singlicate formats. Materials & methods: We employed modeling to assess and quantify the impact of singlicate to cut point factor (CPF) in scenarios with the duplicate precision from 1–20% and the proportion of well-to-well variance to overall assay variance from 0.01–0.90. The impact to CPF by singlicate is marginal if the well-to-well coefficient of variation is <10% and represents <25% of the overall variability. Results & conclusion: The assay parameters including sensitivity, precision, selectivity, drug and target tolerance were comparable between singlicate and duplicate based assays. Our results suggested the minimal impact of singlicate analysis on ADA assay with good duplicate precision. The study provided additional supportive evidence that the singlicate-based analysis is feasible in ADA ligand binding assays.
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Von Leoprechting, Achim, Renate Kumpf, Susanne Menzel, Dominique Reulle, Ralf Griebel, Martin J. Valler, and Frank H. Büttner. "Miniaturization and Validation of a High-Throughput Serine Kinase Assay Using the Alpha Screen Platform." Journal of Biomolecular Screening 9, no. 8 (December 2004): 719–25. http://dx.doi.org/10.1177/1087057104268805.

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Reducing costs while maintaining the highest readout quality is a precept of modern high-throughput screening. Given the trend toward nonradiometric screening platforms, this has been a big challenge for some kinase target classes. Common issues include lowsensitivity, susceptibility to nonspecific interference, or the need for costly reagents. In this study, the authors describe the feasibility ofminiaturization of a serine kinase assay using generic reagents in the Alpha Screen format. They have validated the robustness of this assay in the course of miniaturization from a 35-to 4.375-µ L final assay volume in 384-and 1536-well formats. Within this volume range, they consistently obtained Z• values above 0.5 and have investigated the suitability of these assay formats for measuring compound effects by testing a set of 25 previously identified active compounds. These active compounds were also reliably identified in the miniaturized assay formats. The results presented here show that the Alpha Screen technology permits robust and cost-efficient miniaturization of serine/threonine kinase assays.
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Lee, Hong-Kee, Lionel D. Lewis, Gregory J. Tsongalis, Bernard C. Schur, Paul J. Jannetto, Steven H. Wong, and Kiang-Teck J. Yeo. "Validation of a CYP2D6 Genotyping Panel on the NanoChip Molecular Biology Workstation." Clinical Chemistry 53, no. 5 (May 1, 2007): 823–28. http://dx.doi.org/10.1373/clinchem.2006.081539.

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Abstract Background: CYP2D6 is a highly polymorphic phase I enzyme that metabolizes 20%–25% of clinically used drugs. The objective of this study was to validate a CYP2D6 genotyping assay with the NanoChip® Molecular Biology Workstation. Methods: We genotyped 200 anonymized human DNA samples with the Pyrosequencing® platform at the Medical College of Wisconsin and with the NanoChip platform at Dartmouth Medical School. We compared CYP2D6 genotypes and resolved samples with genotypic discrepancies with the Jurilab CYP2D6 duplication/deletion assay or with traditional DNA sequencing. The Jurilab assay is a long-range PCR assay used to evaluate sequence structures 3′ of the CYP2D7 and CYP2D6 coding regions. For the NanoChip platform, we performed multipad addressing and duplicate runs to test the intra- and intercartridge precision, within- and between-run precision, and reproducibility of the defined genotypes. Results: We used both platforms to genotype all 200 DNA samples for CYP2D6*3, *4, *5, *6, *7, *8, and gene duplication. The 2 methods showed 99.4% concordance in the genotyping results; we found only 8 discrepant genotypes among 1400 DNA analyses. Confirmatory molecular analysis of the discrepant genotypes revealed that the NanoChip assay showed better agreement. The imprecision of the NanoChip method (CV) was 8.9%–17.7%. Conclusions: This validation study of the NanoChip electronic microarray–based CYP2D6 genotyping assay revealed a CV &lt;20% and good concordance with the Pyrosequencing method and a confirmatory sequencing method.
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James-Pemberton, Philip, Urszula Łapińska, Mark Helliwell, Rouslan V. Olkhov, Oliver J. Hedaux, Christopher J. Hyde, and Andrew M. Shaw. "Accuracy and precision analysis for a biophotonic assay of C-reactive protein." Analyst 145, no. 7 (2020): 2751–57. http://dx.doi.org/10.1039/c9an02516b.

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A multiplexed biophotonic assay platform has been developed using the localised particle plasmon in gold nanoparticles assembled in an array and functionalised for two assays: total IgG and C-reactive protein (CRP).
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Filomena, Angela, Anna Guenther, Hannes Planatscher, Francois Topin, Joseph She, Luca Formichella, Laurent Terradot, et al. "Performance of a Multiplex Serological Helicobacter pylori Assay on a Novel Microfluidic Assay Platform." Proteomes 5, no. 4 (October 3, 2017): 24. http://dx.doi.org/10.3390/proteomes5040024.

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Meng, Fanwen, Jacqueline Jonklaas, and Melvin Khee-Shing Leow. "Interconversion of Plasma Free Thyroxine Values from Assay Platforms with Different Reference Intervals Using Linear Transformation Methods." Biology 10, no. 1 (January 11, 2021): 45. http://dx.doi.org/10.3390/biology10010045.

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Clinicians often encounter thyroid function tests (TFT) comprising serum/plasma free thyroxine (FT4) and thyroid stimulating hormone (TSH) measured using different assay platforms during the course of follow-up evaluations which complicates reliable comparison and interpretation of TFT changes. Although interconversion between concentration units is straightforward, the validity of interconversion of FT4/TSH values from one assay platform to another with different reference intervals remains questionable. This study aims to establish an accurate and reliable methodology of interconverting FT4 by any laboratory to an equivalent FT4 value scaled to a reference range of interest via linear transformation methods. As a proof-of-concept, FT4 was simultaneously assayed by direct analog immunoassay, tandem mass spectrometry and equilibrium dialysis. Both linear and piecewise linear transformations proved relatively accurate for FT4 inter-scale conversion. Linear transformation performs better when FT4 are converted from a more accurate to a less accurate assay platform. The converse is true, whereby piecewise linear transformation is superior to linear transformation when converting values from a less accurate method to a more robust assay platform. Such transformations can potentially apply to other biochemical analytes scale conversions, including TSH. This aids interpretation of TFT trends while monitoring the treatment of patients with thyroid disorders.
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43

Meng, Fanwen, Jacqueline Jonklaas, and Melvin Khee-Shing Leow. "Interconversion of Plasma Free Thyroxine Values from Assay Platforms with Different Reference Intervals Using Linear Transformation Methods." Biology 10, no. 1 (January 11, 2021): 45. http://dx.doi.org/10.3390/biology10010045.

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Clinicians often encounter thyroid function tests (TFT) comprising serum/plasma free thyroxine (FT4) and thyroid stimulating hormone (TSH) measured using different assay platforms during the course of follow-up evaluations which complicates reliable comparison and interpretation of TFT changes. Although interconversion between concentration units is straightforward, the validity of interconversion of FT4/TSH values from one assay platform to another with different reference intervals remains questionable. This study aims to establish an accurate and reliable methodology of interconverting FT4 by any laboratory to an equivalent FT4 value scaled to a reference range of interest via linear transformation methods. As a proof-of-concept, FT4 was simultaneously assayed by direct analog immunoassay, tandem mass spectrometry and equilibrium dialysis. Both linear and piecewise linear transformations proved relatively accurate for FT4 inter-scale conversion. Linear transformation performs better when FT4 are converted from a more accurate to a less accurate assay platform. The converse is true, whereby piecewise linear transformation is superior to linear transformation when converting values from a less accurate method to a more robust assay platform. Such transformations can potentially apply to other biochemical analytes scale conversions, including TSH. This aids interpretation of TFT trends while monitoring the treatment of patients with thyroid disorders.
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44

Conde, A. J., E. Salvatierra, O. Podhajcer, L. Fraigi, and R. E. Madrid. "Wound healing assay in a low-cost microfluidic platform." Journal of Physics: Conference Series 477 (December 31, 2013): 012035. http://dx.doi.org/10.1088/1742-6596/477/1/012035.

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45

Park, S. Y., N. Goeken, H. J. Lee, R. Bolan, M. P. Dube, H. Y. Lee, and G. Silvestri. "Developing High-Throughput HIV Incidence Assay with Pyrosequencing Platform." Journal of Virology 88, no. 5 (December 26, 2013): 2977–90. http://dx.doi.org/10.1128/jvi.03128-13.

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46

Sigua, Christopher L., Persis P. Wadia, and Doug Bost. "Platform Independent Multiplex qPCR Research Assay for Chimerism Analysis." Biology of Blood and Marrow Transplantation 19, no. 2 (February 2013): S307. http://dx.doi.org/10.1016/j.bbmt.2012.11.452.

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47

Bova, Michael P., Lan Nguyen, William Wallace, Caroline Garrido, Ying-Zi Xu, Chris Semko, Kelly Cockcroft, Eric Sandberg, and Frederique Bard. "A Label-Free Approach to Identify Inhibitors of α4β7-Mediated Cell Adhesion to MadCAM." Journal of Biomolecular Screening 16, no. 5 (March 15, 2011): 536–44. http://dx.doi.org/10.1177/1087057111399337.

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Traditionally, cell adhesion assays are performed in a manual workstation format using fluorescence-based readouts. Herein, the authors describe a label-free homogeneous assay to identify inhibitors of α4β7 integrin-mediated cell adhesion to its ligand, the mucosal addressin cell adhesion molecule (MadCAM), using the SRU BIND platform. The biosensor is optically based and comprises a subwavelength polymer grating. The assay was validated using standard compounds and an α4 blocking antibody and correlated very closely with the manual assay format when running a battery of test compounds of varying potencies. Cell adhesion was strictly dependent on the presence of divalent cations where Mg2+ was greater than Ca2+ at promoting cell adhesion. This homogeneous and label-free format exhibited low variability with a calculated Z′ of 0.6. In addition to measuring α4β7-mediated 8866 cell adhesion to MadCAM, the authors also demonstrate that this platform can measure adhesion of Jurkat cells expressing α4β1 to the vascular cell adhesion molecule. Thus, the SRU BIND platform is widely applicable to measuring cell adhesion events mediated by other integrins binding to their receptors in an assay format that is amenable to high-throughput screening.
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48

Harrill, Joshua A., Logan J. Everett, Derik E. Haggard, Thomas Sheffield, Joseph L. Bundy, Clinton M. Willis, Russell S. Thomas, Imran Shah, and Richard S. Judson. "High-Throughput Transcriptomics Platform for Screening Environmental Chemicals." Toxicological Sciences 181, no. 1 (February 4, 2021): 68–89. http://dx.doi.org/10.1093/toxsci/kfab009.

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Abstract New approach methodologies (NAMs) that efficiently provide information about chemical hazard without using whole animals are needed to accelerate the pace of chemical risk assessments. Technological advancements in gene expression assays have made in vitro high-throughput transcriptomics (HTTr) a feasible option for NAMs-based hazard characterization of environmental chemicals. In this study, we evaluated the Templated Oligo with Sequencing Readout (TempO-Seq) assay for HTTr concentration-response screening of a small set of chemicals in the human-derived MCF7 cell model. Our experimental design included a variety of reference samples and reference chemical treatments in order to objectively evaluate TempO-Seq assay performance. To facilitate analysis of these data, we developed a robust and scalable bioinformatics pipeline using open-source tools. We also developed a novel gene expression signature-based concentration-response modeling approach and compared the results to a previously implemented workflow for concentration-response analysis of transcriptomics data using BMDExpress. Analysis of reference samples and reference chemical treatments demonstrated highly reproducible differential gene expression signatures. In addition, we found that aggregating signals from individual genes into gene signatures prior to concentration-response modeling yielded in vitro transcriptional biological pathway altering concentrations (BPACs) that were closely aligned with previous ToxCast high-throughput screening assays. Often these identified signatures were associated with the known molecular target of the chemicals in our test set as the most sensitive components of the overall transcriptional response. This work has resulted in a novel and scalable in vitro HTTr workflow that is suitable for high-throughput hazard evaluation of environmental chemicals.
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49

Yang, Shieh Yueh, Jen Leih Wu, Chun Hsi Tso, Fang Huar Ngou, Hsin Yiu Chou, Fan Hua Nan, Herng Er Horng, and Ming Wei Lu. "A novel quantitative immunomagnetic reduction assay for Nervous necrosis virus." Journal of Veterinary Diagnostic Investigation 24, no. 5 (August 1, 2012): 911–17. http://dx.doi.org/10.1177/1040638712455796.

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Rapid, sensitive, and automatic detection platforms are among the major approaches of controlling viral diseases in aquaculture. An efficient detection platform permits the monitoring of pathogen spread and helps to enhance the economic benefits of commercial aquaculture. Nervous necrosis virus (NNV), the cause of viral encephalopathy and retinopathy, is among the most devastating aquaculture viruses that infect marine fish species worldwide. In the present study, a highly sensitive magnetoreduction assay was developed for detecting target biomolecules with a primary focus on NNV antigens. A standard curve of the different NNV concentrations that were isolated from infected Malabar grouper ( Epinephelus malabaricus) was established before experiments were conducted. The test solution was prepared by homogeneous dispersion of magnetic nanoparticles coated with rabbit anti-NNV antibody. The magnetic nanoparticles in the solution were oscillated by magnetic interaction with multiple externally applied, alternating current magnetic fields. The assay’s limit of detection was approximately 2 × 101 TCID50/ml for NNV. Moreover, the immunomagnetic reduction readings for other aquatic viruses (i.e., 1 × 107 TCID50/ml for Infectious pancreatic necrosis virus and 1 × 106.5 TCID50/ml for grouper iridovirus) were below the background noise in the NNV solution, demonstrating the specificity of the new detection platform.
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50

Cho, Eun Jeong, Ashwini K. Devkota, Gabriel Stancu, Ramakrishna Edupunganti, Ginamarie Debevec, Marc Giulianotti, Richard Houghten, Garth Powis, and Kevin N. Dalby. "A Robust and Cost-Effective Luminescent-Based High-Throughput Assay for Fructose-1,6-Bisphosphate Aldolase A." SLAS DISCOVERY: Advancing the Science of Drug Discovery 25, no. 9 (May 28, 2020): 1038–46. http://dx.doi.org/10.1177/2472555220926146.

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Hypoxic solid tumors induce the stabilization of hypoxia-inducible factor 1 alpha (HIF1α), which stimulates the expression of many glycolytic enzymes and hypoxia-responsive genes. A high rate of glycolysis supports the energetic and material needs for tumors to grow. Fructose-1,6-bisphosphate aldolase A (ALDOA) is an enzyme in the glycolytic pathway that promotes the expression of HIF1α. Therefore, inhibition of ALDOA activity represents a potential therapeutic approach for a range of cancers by blocking two critical cancer survival mechanisms. Here, we present a luminescence-based strategy to determine ALDOA activity. The assay platform was developed by integrating a previously established ALDOA activity assay with a commercial NAD/NADH detection kit, resulting in a significant (>12-fold) improvement in signal/background (S/B) compared with previous assay platforms. A screening campaign using a mixture-based compound library exhibited excellent statistical parameters of Z′ (>0.8) and S/B (~20), confirming its robustness and readiness for high-throughput screening (HTS) application. This assay platform provides a cost-effective method for identifying ALDOA inhibitors using a large-scale HTS campaign.
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