Sensitive Fluorescence Polarization Technique for Rapid Screening of α-Synuclein Oligomerization/Fibrillization Inhibitors

Biochemistry ◽  
2007 ◽  
Vol 46 (44) ◽  
pp. 12522-12529 ◽  
Author(s):  
Kelvin C. Luk ◽  
Edward G. Hyde ◽  
John Q. Trojanowski ◽  
Virginia M.-Y. Lee
Keyword(s):  
Fluorescence Polarization ◽  
Rapid Screening ◽  
Polarization Technique

Assay of an antitumor protein, neocarzinostatin, and its antibody by fluorescence polarization.

1978 ◽  
Vol 24 (12) ◽  
pp. 2139-2144 ◽  
Author(s):  
H Maeda
Keyword(s):  
Pharmacokinetic Parameter ◽  
Fluorescence Polarization ◽  
Fluorescein Isothiocyanate ◽  
Apparent Volume ◽  
Volume Of Distribution ◽  
Radial Immunodiffusion ◽  
Antitumor Antibiotic ◽  
Single Radial Immunodiffusion ◽  
Polarization Technique ◽  
Apparent Volume Of Distribution

Abstract I evaluated use of the fluorescence polarization technique to measure neocarzinostatin, a proteinaceous antitumor antibiotic, and its antibody, in serum. The antigen (neocarzinostatin), labeled with fluorescein isothiocyanate, was allowed to interact with its antibody in a cuvet, in the instrument, yielding an increase in the fluorescence polarization value. Antibody content was determined in the presence of a definite amount of the labeled antigen, fluorescence polarization values increasing in parallel with each addition of antibody. Antigen content was determined with a known amount of antibody, which reacted at first with an unknown amount of antigen in samples, followed by addition of a definite amount of the labeled antigen (competition). I used the method to determine a pharmacokinetic parameter, the apparent volume of distribution for neocarzinostatin in rabbits, using drug-injected rabbit sera. I evaluated precision, accuracy, and reproducibility, using various samples or possible interfering substances such as bilirubin and hemoglobin, and also compared results for antigen with those by single radial immunodiffusion assay. The present assay is fast (less than 2 min), sensitive (less than 10 nmol/liter can be detected), and simple (there is no separation step before readout of the results).

High Throughput Determination of BTEX by a One-Step Fluorescence Polarization Immunoassay

2005 ◽  
Vol 2 (3) ◽  
pp. 227 ◽  
Author(s):  
Sergei A. Eremin ◽  
Dietmar Knopp ◽  
Reinhard Niessner ◽  
Ji Youn Hong ◽  
Song-Ja Park ◽  
...  
Keyword(s):  
Fluorescence Polarization ◽  
Limit Of Detection ◽  
Petroleum Products ◽  
Cross Reactivity ◽  
Rapid Screening ◽  
Fluorescence Polarization Immunoassay ◽  
Phenylcarboxylic Acids ◽  
One Step ◽  
Benzene Toluene

Environmental Context.Benzene, toluene, ethylbenzene, and xylenes (BTEX) are used as solvents in paints and coatings and are constituents of petroleum products. BTEX can contaminate air, water or soil and is toxic; benzene, in particular, is a recognized human carcinogen. Most existing methods for detecting BTEX are time-consuming, complicated and very expensive for routine screening. A rapid immunoassay of BTEX is presented that greatly simplifies environmental monitoring of water contamination. Abstract.For the rapid screening of BTEX (benzene, toluene, ethylbenzene, xylenes), a fluorescence polarization immunoassay (FPIA) was developed using the fluorescence polarization analyzer Abbott TDx. Several fluorescence-labelled tracers were synthesized by binding ethylenediamine fluorescein thiocarbamyl (EDF) to various substituted phenylcarboxylic acids. The binding of 27 tracers with two antisera that can be considered as class-specific for BTEX was investigated to select optimal tracer–antibody pairs. Significant differences were found in binding, titer, sensitivity, and assay kinetics. A best pair of anti-BTEX antiserum and EDF-labelled p-tolylacetic acid tracer was selected for FPIA. To simplify the method, an immunocomplex single reagent was prepared to detect BTEX by a one-step FPIA. One-step FPIA is a rapid homogeneous type of immunoassay that has only one pipetting step, does not need separation of free and bound analyte and can be performed at room temperature. The within-run coefficient of variation was ranged between 3.4% and 5.7%. Furthermore, if the measurement can be done at constant temperature, standards for every assay run are unnecessary. Cross-reactivity studies of petroleum compounds and a BTEX mixture indicated that p-xylene was most reactive with a limit of detection (LOD) of 0.22 µg mL−1 in 50 µL of sample. The LOD for toluene and benzene was 2.1 and 11 µg mL−1 respectively. The immunocomplex single reagent has proven to be significantly more stable than the corresponding solutions of antibody and tracer.

scholarly journals Development of a simple and miniaturized sandwich-like fluorescence polarization assay for rapid screening of SARS-CoV-2 main protease inhibitors

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Gangan Yan ◽  
Dongsheng Li ◽  
Yuan Lin ◽  
Zhenghao Fu ◽  
Haiyan Qi ◽  
...  
Keyword(s):  
Fluorescence Polarization ◽  
High Throughput Screening ◽  
Antiviral Agents ◽  
Competitive Inhibitor ◽  
Rapid Screening ◽  
Screening Assay ◽  
Hydrogen Bond Interactions ◽  
Main Protease ◽  
Fluorescence Polarization Assay

Abstract Background Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is highly transmissible and has caused a pandemic named coronavirus disease 2019 (COVID-19), which has quickly spread worldwide. Although several therapeutic agents have been evaluated or approved for the treatment of COVID-19 patients, efficacious antiviral agents are still lacking. An attractive therapeutic target for SARS-CoV-2 is the main protease (Mpro), as this highly conserved enzyme plays a key role in viral polyprotein processing and genomic RNA replication. Therefore, the identification of efficacious antiviral agents against SARS-CoV-2 Mpro using a rapid, miniaturized and economical high-throughput screening (HTS) assay is of the highest importance at the present. Results In this study, we first combined the fluorescence polarization (FP) technique with biotin-avidin system (BAS) to develop a novel and step-by-step sandwich-like FP screening assay to quickly identify SARS-CoV-2 Mpro inhibitors from a natural product library. Using this screening assay, dieckol, a natural phlorotannin component extracted from a Chinese traditional medicine Ecklonia cava, was identified as a novel competitive inhibitor against SARS-CoV-2 Mpro in vitro with an IC50 value of 4.5 ± 0.4 µM. Additionally, dieckol exhibited a high affinity with SARS-CoV-2 Mpro using surface plasmon resonance (SPR) analysis and could bind to the catalytic sites of Mpro through hydrogen-bond interactions in the predicted docking model. Conclusions This innovative sandwich-like FP screening assay enables the rapid discovery of antiviral agents targeting viral proteases, and dieckol will be an excellent lead compound for generating more potent and selective antiviral agents targeting SARS-CoV-2 Mpro.

scholarly journals Design, Synthesis, and Characterization of Tracers and Development of a Fluorescence Polarization Immunoassay for Rapid Screening of 4,4′-Dinitrocarbanilide in Chicken Muscle

Foods ◽  
2021 ◽  
Vol 10 (8) ◽  
pp. 1822
Author(s):  
Qidi Zhang ◽  
Ming Zou ◽  
Wanyu Wang ◽  
Jinyan Li ◽  
Xiao Liang
Keyword(s):  
Fluorescence Polarization ◽  
Limit Of Detection ◽  
Screening Method ◽  
Sample Pretreatment ◽  
Cross Reactivity ◽  
Rapid Screening ◽  
Fluorescence Polarization Immunoassay ◽  
Design Synthesis ◽  
Chicken Muscle ◽  
Edible Tissues

The compound, 4,4′-dinitrocarbanilide (DNC), is the marker residue of concern in edible tissues of broilers fed with diets containing anticoccidial nicarbazin (NIC). In this study, 25 fluorescein-labeled DNC derivatives (tracers) are synthesized and characterized to develop a rapid fluorescence polarization immunoassay (FPIA) for the detection of DNC in chickens using DNC monoclonal antibodies (mAbs). The effect of the tracer structure on the sensitivity of the FPIA is investigated. Our results show that after optimization, the half maximal inhibitory concentrations (IC50) and limit of detection (LOD) of the FPIA in the buffer are 28.3 and 5.7 ng mL−1, respectively. No significant cross-reactivity (CR < 0.89%) with 15 DNC analogues is observed. The developed FPIA is validated for DNC detection in spiked chicken homogenates, and recoveries ranged from 74.2 to 85.8%, with coefficients of variation <8.6%. Moreover, the total time needed for the detection procedure of the FPIA, including sample pretreatment, is <40 min, which has not been achieved in any other immunoassays for DNC from literature. Our results demonstrate that the FPIA developed here is a simple, sensitive, specific, and reproducible screening method for DNC residues in chickens.

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