scholarly journals A Novel Tool for High-Throughput Screening of Granulocyte-Specific Antibodies Using the Automated Flow Cytometric Granulocyte Immunofluorescence Test (Flow-GIFT)

2011 ◽  
Vol 11 ◽  
pp. 302-309 ◽  
Author(s):  
Xuan Duc Nguyen ◽  
Thomas Dengler ◽  
Monika Schulz-Linkholt ◽  
Harald Klüter
Keyword(s):  
High Throughput ◽  
High Throughput Screening ◽  
Blood Donors ◽  
Rapid Screening ◽  
Antibody Detection ◽  
Immunofluorescence Test ◽  
Flow Cytometric ◽  
Reference Methods ◽  
Test Flow ◽  
Granulocyte Antibodies

Transfusion-related acute lung injury (TRALI) is a severe complication related with blood transfusion. TRALI has usually been associated with antibodies against leukocytes. The flow cytometric granulocyte immunofluorescence test (Flow-GIFT) has been introduced for routine use when investigating patients and healthy blood donors. Here we describe a novel tool in the automation of the Flow-GIFT that enables a rapid screening of blood donations. We analyzed 440 sera from healthy female blood donors for the presence of granulocyte antibodies. As positive controls, 12 sera with known antibodies against anti-HNA-1a, -b, -2a; and -3a were additionally investigated. Whole-blood samples from HNA-typed donors were collected and the test cells isolated using cell sedimentation in a Ficoll density gradient. Subsequently, leukocytes were incubated with the respective serum and binding of antibodies was detected using FITC-conjugated antihuman antibody. 7-AAD was used to exclude dead cells. Pipetting steps were automated using the Biomek NXp Multichannel Automation Workstation. All samples were prepared in the 96-deep well plates and analyzed by flow cytometry. The standard granulocyte immunofluorescence test (GIFT) and granulocyte agglutination test (GAT) were also performed as reference methods. Sixteen sera were positive in the automated Flow-GIFT, while five of these sera were negative in the standard GIFT (anti—HNA 3a, n = 3; anti—HNA-1b, n = 1) and GAT (anti—HNA-2a, n = 1). The automated Flow-GIFT was able to detect all granulocyte antibodies, which could be only detected in GIFT in combination with GAT. In serial dilution tests, the automated Flow-GIFT detected the antibodies at higher dilutions than the reference methods GIFT and GAT. The Flow-GIFT proved to be feasible for automation. This novel high-throughput system allows an effective antigranulocyte antibody detection in a large donor population in order to prevent TRALI due to transfusion of blood products.

IMMUNOHEMATOLOGY: Rapid screening of granulocyte antibodies with a novel assay: flow cytometric granulocyte immunofluorescence test

Transfusion ◽  
2009 ◽  
Vol 49 (12) ◽  
pp. 2700-2708 ◽  
Author(s):  
Xuan Duc Nguyen ◽  
Brigitte Flesch ◽  
Ulrich J. Sachs ◽  
Hartmut Kroll ◽  
Harald Klüter ◽  
...  
Keyword(s):  
Rapid Screening ◽  
Immunofluorescence Test ◽  
Flow Cytometric ◽  
Granulocyte Antibodies

Development of a dual-fluorescence reporter system for high-throughput screening of L-aspartate-α-decarboxylase

2020 ◽  
Vol 52 (12) ◽  
pp. 1420-1426
Author(s):  
Mingyue Fei ◽  
Xudan Mao ◽  
Yiyang Chen ◽  
Yalan Lu ◽  
Lin Wang ◽  
...  
Keyword(s):  
High Throughput ◽  
Industrial Production ◽  
High Throughput Screening ◽  
Relative Activity ◽  
Expression Level ◽  
Rapid Screening ◽  
Dual Fluorescence ◽  
Reporter System ◽  
The Cost ◽  
Chemical Synthesis Route

Abstract β-Alanine (3-aminopropionic acid) holds great potential in industrial application. It can be obtained through a chemical synthesis route, which is hazardous to the environment. It is well known that l-aspartate-α-decarboxylase (ADC) can convert l-aspartate to β-alanine in bacteria. However, due to the low activity of ADC, industrial production of β-alanine through the green biological route remains unclear. Thus, improving the activity of ADC is critical to reduce the cost of β-alanine production. In this study, we established a dual-fluorescence high-throughput system for efficient ADC screening. By measuring the amount of β-alanine and the expression level of ADC using two different fluorescence markers, we can rapidly quantify the relative activity of ADC variants. From a mutagenesis library containing 2000 ADC variants, we obtained a mutant with 33% increased activity. Further analysis revealed that mutations of K43R and P103Q in ADC significantly improved the yield of β-alanine produced by the whole-cell biocatalysis. Compared with the previous single-fluorescence method, our system can not only quantify the amount of β-alanine but also measure the expression level of ADC with different fluorescence, making it able to effectively screen out ADC variants with improved relative activity. The dual-fluorescence high-throughput system for rapid screening of ADC provides a good strategy for industrial production of β-alanine via the biological conversion route in the future.

scholarly journals Recovery of NanoLuc Luciferase-Tagged Canine Distemper Virus for Facilitating Rapid Screening of Antivirals in vitro

2020 ◽  
Vol 7 ◽  
Author(s):  
Fuxiao Liu ◽  
Qianqian Wang ◽  
Yilan Huang ◽  
Ning Wang ◽  
Youming Zhang ◽  
...  
Keyword(s):  
High Throughput ◽  
High Throughput Screening ◽  
Canine Distemper Virus ◽  
Reverse Genetics ◽  
Rapid Screening ◽  
Canine Distemper ◽  
Virus Propagation ◽  
The Family ◽  
Conventional Methods

Canine distemper virus (CDV), belonging to the genus Morbillivirus in the family Paramyxoviridae, is a highly contagious pathogen, affecting various domestic, and wild carnivores. Conventional methods are too cumbersome to be used for high-throughput screening of anti-CDV drugs. In this study, a recombinant CDV was rescued using reverse genetics for facilitating screening of anti-CDV drug in vitro. The recombinant CDV could stably express the NanoLuc® luciferase (NLuc), a novel enzyme that was smaller and “brighter” than others. The intensity of NLuc-catalyzed luminescence reaction indirectly reflected the anti-CDV effect of a certain drug, due to a positive correlation between NLuc expression and virus propagation in vitro. Based on such a characteristic feature, the recombinant CDV was used for anti-CDV assays on four drugs (ribavirin, moroxydine hydrochloride, 1-adamantylamine hydrochloride, and tea polyphenol) via analysis of luciferase activity, instead of via conventional methods. The result showed that out of these four drugs, only the ribavirin exhibited a detectable anti-CDV effect. The NLuc-tagged CDV would be a rapid tool for high-throughput screening of anti-CDV drugs.

scholarly journals Optimization of a High-Throughput 384-Well Plate-Based Screening Platform with Staphylococcus aureus ATCC 25923 and Pseudomonas aeruginosa ATCC 15442 Biofilms

2020 ◽  
Vol 21 (9) ◽  
pp. 3034 ◽  
Author(s):  
Shella Gilbert-Girard ◽  
Kirsi Savijoki ◽  
Jari Yli-Kauhaluoma ◽  
Adyary Fallarero
Keyword(s):  
Staphylococcus Aureus ◽  
Pseudomonas Aeruginosa ◽  
High Throughput ◽  
High Throughput Screening ◽  
Bacterial Infections ◽  
Rapid Screening ◽  
Bacterial Biofilms ◽  
Main Concern ◽  
Assay Optimization ◽  
Screening Platform

In recent years, bacterial infections have become a main concern following the spread of antimicrobial resistance. In addition, bacterial biofilms are known for their high tolerance to antimicrobials and they are regarded as a main cause of recalcitrant infections in humans. Many efforts have been deployed in order to find new antibacterial therapeutic options and the high-throughput screening (HTS) of large libraries of compounds is one of the utilized strategies. However, HTS efforts for anti-biofilm discovery remain uncommon. Here, we miniaturized a 96-well plate (96WP) screening platform, into a 384-well plate (384WP) format, based on a sequential viability and biomass measurements for the assessment of anti-biofilm activity. During the assay optimization process, different parameters were evaluated while using Staphylococcus aureus and Pseudomonas aeruginosa as the bacterial models. We compared the performance of the optimized 384WP platform to our previously established 96WP-based platform by carrying out a pilot screening of 100 compounds, followed by the screening of a library of 2000 compounds to identify new repurposed anti-biofilm agents. Our results show that the optimized 384WP platform is well-suited for screening purposes, allowing for the rapid screening of a higher number of compounds in a run in a reliable manner.

scholarly journals A Novel High-Throughput Screening Assay for Sickle Cell Disease Drug Discovery

2009 ◽  
Vol 14 (4) ◽  
pp. 330-336 ◽  
Author(s):  
Eszter Pais ◽  
John S. Cambridge ◽  
Cage S. Johnson ◽  
Herbert J. Meiselman ◽  
Timothy C. Fisher ◽  
...  
Keyword(s):  
Sickle Cell Disease ◽  
Sickle Cell ◽  
High Throughput ◽  
High Throughput Screening ◽  
Rapid Screening ◽  
Cell Disease ◽  
Screening Assay ◽  
Drug Candidates ◽  
Test Molecule ◽  
High Throughput Screening Assay

Although the pathophysiology and molecular basis of sickle cell disease (SCD) were described more than half a century ago, an effective and safe therapy is not yet available. This may be explained by the lack of a suitable high-throughput technique that allows rapid screening of thousands of compounds for their antisickling effect. The authors have thus developed a novel high-throughput screening (HTS) assay based on detecting the ability of red blood cells (RBC) to traverse a column of tightly packed Sephacryl chromatography beads. When deoxygenated, sickle RBC are rigid and remain on the top of the column. However, when deoxygenated and treated with an effective antisickling agent, erythrocytes move through the Sephacryl media and produce a red dot on the bottom of the assay tubes. This approach has been adapted to wells in a 384-well microplate. Results can be obtained by optical scanning: The size of the red dot is proportional to the antisickling effect of the test molecule. The new assay is simple, inexpensive, reproducible, requires no special reagents, and should be readily adaptable to robotic HTS systems. It has the potential to identify novel drug candidates, allowing the development of new therapeutic options for individuals affected with SCD. ( Journal of Biomolecular Screening. 2009:330-336)

scholarly journals Rapid, noninvasive, and unsupervised detection of sleep/wake using piezoelectric monitoring for pharmacological studies in narcoleptic mice

2017 ◽  
Author(s):  
Sarah Wurts Black ◽  
Jessica D. Sun ◽  
Alex Laihsu ◽  
Nikki Kimura ◽  
Pamela Santiago ◽  
...  
Keyword(s):  
Drug Discovery ◽  
High Throughput ◽  
High Throughput Screening ◽  
Body Movement ◽  
Rapid Screening ◽  
Wild Type ◽  
Preclinical Development ◽  
Pharmacological Studies ◽  
Dose Response Study

AbstractBackgroundAssessment of sleep/wake by electroencephalography (EEG) and electromyography (EMG) is invasive, resource intensive, and not amenable to rapid screening at scale for drug discovery. In the preclinical development of therapeutics for narcolepsy, efficacy tests are hindered by the lack of a non-EEG/EMG based translational test of symptom severity. The current methods study offers proof-of-principle that PiezoSleep (noninvasive, unsupervised piezoelectric monitoring of gross body movement, together with respiration patterns during behavioral quiescence), can be used to determine sleep/wake as applicable to the development of wake-promoting therapeutics. First, the translational wake-maintenance score (WMS, the ratio of time during the first half of the dark period spent in long wake bouts to short sleep bouts) of the PiezoSleep narcolepsy screen was introduced as a means by which to rank narcoleptic orexin/ataxin-3 mice and wild type mice by sleep/wake fragmentation severity. Accuracy of the WMS to detect narcoleptic phenotypes were determined in genotype-confirmed orexin/ataxin-3 mice and wild type colony mates. The WMS was used to identify the most highly symptomatic mice for resource-intensive EEG/EMG studies for further analysis of specific arousal states. Second, PiezoSleep was demonstrated for use in high-throughput screening of wake-promoting compounds using modafinil in orexin/ataxin-3 and wild type mice.ResultsThe WMS detected a narcoleptic phenotype with 89% sensitivity, 92% specificity and 98% positive predictive value. A 15-fold difference in WMS differentiated wild type littermates from the most severely affected orexin/ataxin-3 mice. Follow-up EEG/EMG study indicated 82% of the orexin/ataxin-3 mice with the lowest wake-maintenance scores met or exceeded the cataplexy-occurrence threshold (≥ 3 bouts) for inclusion in therapeutic efficacy studies. In the PiezoSleep dose-response study, the ED50 for wake-promotion by modafinil was approximately 50 mg/kg in both genotypes. Using unsupervised piezoelectric monitoring, the efficacy of wake-promoting compounds can be determined in a 5-arm study with 60 mice in less than one week—a fraction of the time compared to EEG/EMG studies.ConclusionsThe WMS on the PiezoSleep narcolepsy screen quantifies the inability to sustain wakefulness and provides an accurate measure of the narcoleptic phenotype in mice. PiezoSleep offers rapid, scalable assessment of sleep/wake for high-throughput screening in drug discovery.

scholarly journals Improved Statistical Methods for Hit Selection in High-Throughput Screening

2003 ◽  
Vol 8 (6) ◽  
pp. 634-647 ◽  
Author(s):  
Christine Brideau ◽  
Bert Gunter ◽  
Bill Pikounis ◽  
Andy Liaw
Keyword(s):  
Data Analysis ◽  
Statistical Methods ◽  
High Throughput ◽  
High Throughput Screening ◽  
Drug Targets ◽  
Software Tool ◽  
Rapid Screening ◽  
Modern Drug ◽  
Server Software ◽  
Technical Details

High-throughput screening (HTS) plays a central role in modern drug discovery, allowing the rapid screening of large compound collections against a variety of putative drug targets. HTS is an industrial-scale process, relying on sophisticated auto mation, control, and state-of-the art detection technologies to organize, test, and measure hundreds of thousands to millions of compounds in nano-to microliter volumes. Despite this high technology, hit selection for HTS is still typically done using simple data analysis and basic statistical methods. The authors discuss in this article some shortcomings of these methods and present alternatives based on modern methods of statistical data analysis. Most important, they describe and show numerous real examples from the biologist-friendly Stat Server® HTS application (SHS), a custom-developed software tool built on the commercially available S-PLUS® and StatServer® statistical analysis and server software. This system remotely processes HTS data using powerful and sophisticated statistical methodology but insulates users from the technical details by outputting results in a variety of readily interpretable graphs and tables.

High-throughput screening of bimetallic catalysts enabled by machine learning

2017 ◽  
Vol 5 (46) ◽  
pp. 24131-24138 ◽  
Author(s):  
Zheng Li ◽  
Siwen Wang ◽  
Wei Shan Chin ◽  
Luke E. Achenie ◽  
Hongliang Xin
Keyword(s):  
Machine Learning ◽  
Kinetic Analysis ◽  
High Throughput ◽  
Bimetallic Catalysts ◽  
High Throughput Screening ◽  
Rapid Screening ◽  
Learning Framework

We present a holistic machine-learning framework for rapid screening of bimetallic catalysts with the aid of the descriptor-based kinetic analysis.

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