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A rapid, facile and selective detection of anti-H5 subtype avian influenza virus (AIV) antibody in serum by fluorescence polarization immunoassay (FPIA) was achieved. A fragment of recombinant H5 subtype AIV hemagglutinin was produced and labeled with fluorescein to use it as a labeled antigen in FPIA. This labeled antigen was mixed with anti-AIV sera (H1-H16 subtypes) and FP of the mixture was measured using a portable FP analyzer on a microdevice. It was found that FP increased in proportion to the concentration of anti-H5 AIV antibody (serum) and was significantly higher than FP obtained with the other sera. The selective detection of anti-H5 subtype AIV antibody was confirmed. The required volume of original sample was 2 μL and analysis time was within 20 min. This detection system realizes an efficient on-site diagnosis and surveillance of AIV.
Prompt diagnosis, patient isolation, and contact tracing are key measures to contain the coronavirus disease 2019 (COVID-19). Molecular tests are the current gold standard for COVID-19 detection, but are carried out at central laboratories, delaying treatment and control decisions. Here we describe a portable assay system for rapid, onsite COVID-19 diagnosis. Termed CODA (CRISPR Optical Detection of Anisotropy), the method combined isothermal nucleic acid amplification, activation of CRISPR/Cas12a, and signal generation in a single assay, eliminating extra manual steps. Importantly, signal detection was based on the ratiometric measurement of fluorescent anisotropy, which allowed CODA to achieve a high signal-to-noise ratio. For point-of-care operation, we built a compact, standalone CODA device integrating optoelectronics, an embedded heater, and a microcontroller for data processing. The developed system completed SARS-CoV-2 RNA detection within 20 min of sample loading; the limit of detection reached 3 copy/μL. When applied to clinical samples (10 confirmed COVID-19 patients; 10 controls), the rapid CODA test accurately classified COVID-19 status, in concordance with gold-standard clinical diagnostics.
Protein tyrosine phosphatases (PTPs) are important signaling enzymes that control such fundamental processes as proliferation, differentiation, survival/apoptosis, as well as adhesion and motility. Potent and selective PTP inhibitors serve not only as powerful research tools, but also as potential therapeutics against a variety illness including cancer and diabetes. PTP activity-based assays are widely used in high throughput screening (HTS) campaigns for PTP inhibitor discovery. These assays suffer from a major weakness, in that the reactivity of the active site Cys can cause serious problems as highly reactive oxidizing and alkylating agents may surface as hits. We describe the development of a fluorescence polarization (FP)-based displacement assay that makes the use of an active site Cys to Ser mutant PTP (e.g., PTP1B/C215S) that retains the wild-type binding affinity. The potency of library compounds is assessed by their ability to compete with the fluorescently labeled active site ligand for binding to the Cys to Ser PTP mutant. Finally, the substitution of the active site Cys by a Ser renders the mutant PTP insensitive to oxidation and alkylation and thus will likely eliminate "false" positives due to modification of the active site Cys that destroy the phosphatase activity.
Pharmacologically active compounds are often detected in wastewater and surface waters. The nonsteroidal anti-inflammatory drug diclofenac (DCF) was included in the European watch list of substances that requires its environmental monitoring in the member states. DCF may harmfully influence the ecosystem already at concentrations ≤ 1 μg L-1. The fast and easy quantification of DCF is becoming a subject of global importance. Fluorescence polarization immunoassay (FPIA) is a homogeneous mix-and-read method which does not require the immobilization of reagents. FPIA can be performed in one phase within 20-30 min, making it possible to analyse wastewater without any complicated pre-treatment. In this study, new tracer molecules with different structures, linking fluorophores to derivatives of the analyte, were synthesized, three homologous tracers based on DCF, two including a C6 spacer, and one heterologous tracer derived from 5-hydroxy-DCF. The tracer molecules were thoroughly assessed for performance. Regarding sensitivity of the FPIA, the lowest limit of detection reached was 2.0 μg L-1 with a working range up to 870 μg L-1. The method was validated for real wastewater samples against LC-MS/MS as reference method with good agreement of both methods. Graphical abstract.
Detection of ciprofloxacin residues in milk by sensitive and rapid methods is of great interest due to its use in the treatment of dairy livestock health. Current analytical approaches to antibiotics detection, are laboratory-based methods and they are time-consuming and require trained personnel. To cope this problem, we propose an assay, based on fluorescence polarization principle, able to detect the presence of ciprofloxacin in diluted milk sample without any pre-treatment. The proposed method is based on the use of ciprofloxacin-protein conjugate labeled with near infrared fluorescence dye, which upon binding to specific antibody causes an increase of the fluorescence polarization emission signal. The developed assay allows for the detection of ciprofloxacin at a concentration of 1ppb, which represents an amount lower than the maximum residual limit (MRL) of ciprofloxacin in milk, as set by the European Union regulation (100 ppb).
Dibutyl phthalate (DBP) is widely used as a plasticizer in the production of polymeric materials to give them flexibility, strength and extensibility. However, due to its negative impact on human health, in particular reproductive functions and fetal development, the content of DBP must be controlled in food and the environment. The present study aims to develop a sensitive, fast and simple fluorescence polarization immunoassay (FPIA) using monoclonal antibodies derived against DBP (MAb-DBP) for its detection in open waters. New conjugates of DBP with various fluorescein derivatives were obtained and characterized: 5-aminomethylfluorescein (AMF) and dichlorotriazinylaminofluorescein (DTAF). The advantages of using the DBP-AMF conjugate in the FPIA method are shown, the kinetics of binding of this chemical with antibodies are studied, the analysis is optimized, and the concentration of monoclonal antibodies is selected for sensitivity analysis-16 nM. The calibration dependence of the fluorescence polarization signal for the detection of DBP was obtained. The observed IC50 (DBP concentration at which a 50% decrease in the fluorescence polarization signal occurs, 40 ng/mL) and the limit of detection (LOD, 7.5 ng/mL) values were improved by a factor of 45 over the previously described FPIA using polyclonal antibodies. This technique was tested by the recovery method, and the high percentage of DBP discovery in water ranged from 85 to 110%. Using the developed method, real water samples from Lake Onega were tested, and a good correlation was shown between the results of the determination of DBP by the FPIA method and GC-MS. Thus, the FPIA method developed in this work can be used to determine DBP in open-water reservoirs.
Developmental, homeostatic, and pharmacological pro-apoptotic signals converge by activating the BCL-2 family member BAX. Studies investigating molecular regulation of BAX are commonly limited to methodologies measuring endpoint phenotypes and do not assess activation of monomeric BAX. Here, we present FLAMBE, a fluorescence polarization ligand assay for monitoring BAX early activation, that measures activation-induced release of a peptide probe in real time. Using complementary parallel and tandem biochemical techniques, we validate, corroborate, and apply FLAMBE to a contemporary repertoire of BAX modulators, characterizing their contributions within the early steps of BAX activation. Additionally, we use FLAMBE to reveal that historically "dead" BAX mutants remain responsive to activation as quasi-functional monomers. We also identify data metrics for comparative analyses and demonstrate that FLAMBE data align with downstream functional observations. Collectively, FLAMBE advances our understanding of BAX activation and fills a methodological void for studying BAX with broad applications in cell biology and therapeutic development. MOTIVATION In vitro BAX activation studies are invaluable platforms for studying cellular and pharmacological modulators of apoptosis. The gold standard for studying BAX function relies on membrane permeabilization assays, which assess the pore-forming activity of oligomeric BAX. However, there are currently no rapid or kinetic assays to interrogate real-time activation of monomeric BAX in solution, thereby limiting any molecular insights that occur upstream of mitochondrial permeabilization. Furthermore, available methods to observe the activation of monomeric BAX suffer from low throughput and static observations. To address this methodological gap, we developed FLAMBE, a kinetic fluorescence polarization-based assay to measure monomeric BAX activation in solution via concomitant displacement of a labeled peptide. This approach maintains the benefits of rapid kinetic data generation in a low-cost microplate format without requiring specialized equipment or large quantities of protein. FLAMBE compliments available experimental strategies and expands the accessibility of investigators to monitor early steps within the BAX activation continuum.
A fluorescence polarization immunoassay (FPIA) for the determination of imidacloprid (IMI) was developed with advantages of simple operation and short assay time. The haptens of IMI, acetamiprid (ACE), and thiamethoxam (THI) were conjugated with fluorescein isothiocyanate ethylenediamine (EDF) and 4'-Aminomethyl fluorescein (AMF), respectively, to prepare six fluorescence tracers. The conjugation of IMI hapten and EDF (IMI-EDF) was selected to develop the FPIA due to the largest fluorescent polarization value increase in the presence of anti-IMI monoclonal antibody. Under the optimum condition, the limit of detection, 50% inhibition concentration and detection range of the FPIA were 1.7, 4.8, and 1.7-16.3 μg/L, respectively. The cross-reactivities (CRs) with the analogs of IMI were negligible except for imidaclothiz with CR of 79.13%. The average recovery of spiked paddy water, corn and cucumber samples were 82.4-118.5% with the RSDs of 7.0-15.9%, which indicated the FPIA had good accuracy. Thus, the developed FPIA was a potential tool for the rapid and accurate determination of IMI in agricultural and environmental samples.
Oxygen-dependent ubiquitination and degradation of hypoxia-inducible factor 1alpha (HIF-1alpha) plays a central role in regulating transcriptional responses to hypoxia. This process requires hydroxylation of specific prolines in HIF-1alpha by HIF prolyl hydroxylase domain (PHD)-containing enzymes, leading to its specific interactions with von Hippel-Lindau protein-Elongin B-Elongin C (VBC). Here we describe a straightforward approach to apply these interactions to measure PHD activities. Employing fluorescently labeled HIF-1alpha peptides containing hydroxyproline, we developed a quantitative method based on fluorescence polarization for a systematic evaluation of binding of hydroxylated HIF-1alpha to recombinant VBC. The method was then successfully utilized for measuring the activity of the truncated, purified PHD2. The applicability of the assay was further demonstrated by examining effects of various cofactors and inhibitors for PHD2. The developed homogeneous assay would provide a convenient way of probing the biochemical properties of the HIF-1alpha-VBC interaction and PHDs, and of screening modulators for the interaction as well as the enzyme.
Brucellosis is an infectious-allergic zoonotic disease caused by bacteria of the genus Brucella. Early diagnosis is the key to preventing, treating, and controlling brucellosis. Fluorescence polarization immunoassay (FPA) is a new immunoassay for relatively rapid and accurate detection of antibodies or antigens based on antigen-antibody interaction. However, there is no report on FPA-based detection of human brucellosis in China. Therefore, this study is to evaluate the value of FPA for the diagnosis of human brucellosis in China.
Aspergillus fumigatus is an opportunistic human pathogenic fungus responsible for deadly lung infections in immunocompromised individuals. Galactofuranose (Galf) residues are essential components of the cell wall and play an important role in A. fumigatus virulence. The flavoenzyme UDP-galactopyranose mutase (UGM) catalyzes the isomerization of UDP-galactopyranose to UDP-galactofuranose, the biosynthetic precursor of Galf. Thus, inhibitors of UGM that block the biosynthesis of Galf can lead to novel chemotherapeutics for treating A. fumigatus-related diseases. Here, we describe the synthesis of fluorescently labeled UDP analogs and the development of a fluorescence polarization (FP) binding assay for A. fumigatus UGM (AfUGM). High-affinity binding to AfUGM was only obtained with the chromophore TAMRA, linked to UDP by either 2 or 6 carbons with K(d) values of 2.6 ± 0.2 μM and 3.0 ± 0.7 μM, respectively. These values were ~6 times lower than when UDP was linked to fluorescein. The FP assay was validated against several known ligands and displayed an excellent Z' factor (0.79 ± 0.02) and good tolerance to dimethyl sulfoxide.
BAX activation techniques are crucial to studying the intrinsic pathway of apoptosis- thousands of pro-apoptotic signals converge on BAX activation. Current methodologies are predominantly limited to membrane permeabilization studies, which assess endpoint functionality of oligomeric BAX, but overlook early activation steps of cytosolic BAX. Here we detail FLAMBE: a fluorescence polarization ligand assay for monitoring BAX early-activation in solution. We also describe a dual-metric parameterization strategy for distillation of kinetic data and comparative analyses when studying candidate ligands. For complete details on the use and execution of this protocol, please refer to Gelles et al. (2022).
Adiponectin, the adipose-derived hormone, plays an important role in the suppression of metabolic disorders that can result in type 2 diabetes, obesity, and atherosclerosis. It has been shown that up-regulation of adiponectin or adiponectin receptor has a number of therapeutic benefits. Given that it is hard to convert the full size adiponectin protein into a viable drug, adiponectin receptor agonists could be designed or identified using high-throughput screening. Here, we report on the development of a two-step screening process to identify adiponectin agonists. First step, we developed a high throughput screening assay based on fluorescence polarization to identify adiponectin ligands. The fluorescence polarization assay reported here could be adapted to screening against larger small molecular compound libraries. A natural product library containing 10,000 compounds was screened and 9 hits were selected for validation. These compounds have been taken for the second-step in vitro tests to confirm their agonistic activity. The most active adiponectin receptor 1 agonists are matairesinol, arctiin, (-)-arctigenin and gramine. The most active adiponectin receptor 2 agonists are parthenolide, taxifoliol, deoxyschizandrin, and syringin. These compounds may be useful drug candidates for hypoadiponectin related diseases.
As a widely used neonicotinoid insecticide, thiacloprid has been observed to pose a risk to honeybees and the endocrine system of mammals. So a detection method with high sensitivity, simple operation and high throughput is required. Based on this consideration, we prepared an anti-thiacloprid monoclonal antibody (mAb, C9) and developed a fluorescence polarization immunoassay (FPIA) for the detection of thiacloprid. After optimizing the length of spacer and reaction conditions, the 50% inhibition concentration (IC50), limit of detection (LOD) and linear range (IC20 ∼ IC80) of the FPIA are 15.34 ng mL-1, 2.43 ng mL-1 and 3.10-65.7 ng mL-1, respectively. Meanwhile, FPIA just requires 12 min to detect the pesticide with simple operation. Then the FPIA was used to detect the thiacloprid in spiked rice, soil, cucumber and tomato samples, and recoveries were in the range of 79.1%-105.3% with 3.7%-12.3% standard deviation. The FPIA also shows good correlation with high-performance liquid chromatography for the detection of thiacloprid in tomato samples.
The ABL protein-tyrosine kinase regulates intracellular signaling pathways controlling diverse cellular processes and contributes to several forms of cancer. The kinase activity of ABL is repressed by intramolecular interactions involving its regulatory Ncap, SH3 and SH2 domains. Small molecules that allosterically regulate ABL kinase activity through its non-catalytic domains may represent selective probes of ABL function. Here we report a screening assay for chemical modulators of ABL kinase activity that target the regulatory interaction of the SH3 domain with the SH2-kinase linker. This fluorescence polarization (FP) assay is based on a purified recombinant ABL protein consisting of the N-cap, SH3 and SH2 domains plus the SH2-kinase linker (N32L protein) and a short fluorescein-labeled probe peptide that binds to the SH3 domain. In assay development experiments, we found that the probe peptide binds to the recombinant ABL N32L protein in vitro, producing a robust FP signal that can be competed with an excess of unlabeled peptide. The FP signal is not observed with control N32L proteins bearing either an inactivating mutation in the SH3 domain or enhanced SH3:linker interaction. A pilot screen of 1200 FDA-approved drugs identified four compounds that specifically reduced the FP signal by at least three standard deviations from the untreated controls. Secondary assays showed that one of these hit compounds, the antithrombotic drug dipyridamole, enhances ABL kinase activity in vitro to a greater extent than the previously described ABL agonist, DPH. Docking studies predicted that this compound binds to a pocket formed at the interface of the SH3 domain and the linker, suggesting that it activates ABL by disrupting this regulatory interaction. These results show that screening assays based on the non-catalytic domains of ABL can identify allosteric small molecule regulators of kinase function, providing a new approach to selective drug discovery for this important kinase system.
Förster resonance energy transfer (FRET) microscopy is frequently used to study protein interactions and conformational changes in living cells. The utility of FRET is limited by false positive and negative signals. To overcome these limitations we have developed Fluorescence Polarization and Fluctuation Analysis (FPFA), a hybrid single-molecule based method combining time-resolved fluorescence anisotropy (homo-FRET) and fluorescence correlation spectroscopy. Using FPFA, homo-FRET (a 1-10 nm proximity gauge), brightness (a measure of the number of fluorescent subunits in a complex), and correlation time (an attribute sensitive to the mass and shape of a protein complex) can be simultaneously measured. These measurements together rigorously constrain the interpretation of FRET signals. Venus based control-constructs were used to validate FPFA. The utility of FPFA was demonstrated by measuring in living cells the number of subunits in the α-isoform of Venus-tagged calcium-calmodulin dependent protein kinase-II (CaMKIIα) holoenzyme. Brightness analysis revealed that the holoenzyme has, on average, 11.9 ± 1.2 subunit, but values ranged from 10-14 in individual cells. Homo-FRET analysis simultaneously detected that catalytic domains were arranged as dimers in the dodecameric holoenzyme, and this paired organization was confirmed by quantitative hetero-FRET analysis. In freshly prepared cell homogenates FPFA detected only 10.2 ± 1.3 subunits in the holoenzyme with values ranging from 9-12. Despite the reduction in subunit number, catalytic domains were still arranged as pairs in homogenates. Thus, FPFA suggests that while the absolute number of subunits in an auto-inhibited holoenzyme might vary from cell to cell, the organization of catalytic domains into pairs is preserved.
Ochratoxin A (OTA) is a known food contaminant that affects a wide range of food and agricultural products. The presence of this fungal metabolite in foods poses a threat to human health. Therefore, various detection and quantification methods have been developed to determine its presence in foods. Herein, we describe a rapid and ultrasensitive tracer-based fluorescence polarization immunoassay (FPIA) for the detection of OTA in rice samples. Four fluorescent tracers OTA-fluorescein thiocarbamoyl ethylenediamine (EDF), OTA-fluorescein thiocarbamoyl butane diamine (BDF), OTA-amino-methyl fluorescein (AMF), and OTA-fluorescein thiocarbamoyl hexame (HDF) with fluorescence polarization values (δFP = FPbind-FPfree) of 5, 100, 207, and 80 mP, respectively, were synthesized. The tracer with the highest δFP value (OTA-AMF) was selected and further optimized for the development of an ultrasensitive FPIA with a detection range of 0.03-0.78 ng/mL. A mean recovery of 70.0% to 110.0% was obtained from spiked rice samples with a relative standard deviation of equal to or less than 20%. Good correlations (r2 = 0.9966) were observed between OTA levels in contaminated rice samples obtained by the FPIA method and high-performance liquid chromatography (HPLC) as a reference method. The rapidity of the method was confirmed by analyzing ten rice samples that were analyzed within 25 min, on average. The sensitivity, accuracy, and rapidity of the method show that it is suitable for screening and quantification of OTA in food samples without the cumbersome pre-analytical steps required in other mycotoxin detection methods.
The AcrB of Escherichia coli pumps out a wide range of compounds, including most of the currently available antibiotics, and contributes significantly to the serious problem of multidrug resistance of pathogenic bacteria. Quantitative analysis of drug efflux by this pump requires the measurement of the affinity of ligands. Yet there has been no success in determining these values. We introduce here an approach of steady-state fluorescence polarization to study the interactions between four different ligands and the purified AcrB transporter in a detergent environment. Our assays indicate that the transporter binds these drugs with K(D) values ranging from 5.5 to 74.1microM.
We recently characterized the association between DNA damage and immunoresponse in vivo in colonic mucosa of mice infected with a Salmonella Typhimurium strain expressing a genotoxin, known as typhoid toxin. In this protocol, we describe how to assess the extent and features of infiltrating macrophages by double immunofluorescence. Total macrophage population was determined using an F4/80 antibody, whereas the specific M2-like population was assessed using a CD206 antibody. For complete details on the use and execution of this protocol, please refer to Martin et al. (2021).
The primary goal of cancer surgery is to completely eliminate tumors. A real-time diagnostic method uses a fluorescence contrast agent and a surgical microscope to assess the status of tumor resection and the patient's blood circulation. The biggest problem in imaging diagnostics using a microscope is the specular reflection phenomenon. While observing a lesion, the observation field may be obstructed due to specular reflection, making it difficult to obtain accurate results during the diagnostic process. Herein we propose a method to reduce specular reflection during tumor diagnosis by introducing a linearly polarized filter for a surgical microscope system. The method of angular direction adjustment of the filter ensures that only the horizontally polarized light passes through it, thereby obstructing the specular reflection. As a result of removing specular reflection, clear images were obtained at 90° and 270°. This experiment was conducted using phantoms and animals. Our results prove that the proposed method can be applied to imaging cameras used in internal medicine, surgery, and radiology for diagnosis.
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