Non-competitive fluorescence polarization immunoassay for detection of H5 avian influenza virus using a portable analyzer.

Nowadays, the diagnosis of viral infections is receiving broad attention. We have developed a non-competitive fluorescence polarization immunoassay (NC-FPIA), which is a separation-free immunoassay, for a virus detection. H5 subtype avian influenza virus (H5-AIV) was used as a model virus for the proof of concept. The fluorescein-labeled Fab fragment that binds to H5 hemagglutinin was used for NC-FPIA. The purified H5-AIV which has H5 hemagglutinin was mixed with the fluorescein-labeled Fab fragment. After that, the degree of fluorescence polarization was measured with a portable FPIA analyzer. H5-AIV was successfully detected with an incubation time of 15 min. In addition, the portable FPIA analyzer enables performance of on-site NC-FPIA with a sample volume of 20 µL or less. This is the first research of detecting a virus particle by FPIA. This NC-FPIA can be applied to rapid on-site diagnosis of various viruses.

Screening of several drugs of abuse in Italian workplace drug testing: performance comparisons of on-site screening tests and a fluorescence polarization immunoassay-based device.

According to the Italian laws, some categories of workers entrusted with duties possibly constituting a threat to security, physical safety, and health of third parties have to be screened to exclude the use/abuse of the following drugs of abuse: opiates, cocaine, cannabinoids, amphetamine, methamphetamine, 3,4-methylenedioxymethamphetamine, methadone, and buprenorphine. Toxicological tests can be performed with urinary on-site rapid screening devices, provided that sensitivities up to specified cutoffs are ensured. The present study reports performances, in terms of sensitivity, specificity, and accuracy, of an automatic on-site test and of an FPIA-based device, using gas chromatography/mass spectrometry (GC/MS) as a reference methodology. Three levels of concentration were tested, corresponding to the cutoff and to 2 and 3 times the limits, respectively. In terms of sensitivities, neither the on-site nor the benchtop instrumentations gave positive results, since values of zero percentage were obtained for concentrations up to 2-fold the limits. Even if good results were obtained in terms of specificity and accuracy by both devices, none of them seem to be adequate for the current application to the toxicological screening at workplaces. In fact, a rapid screening device can be used for drug tests provided that it ensures sensitivity at the prescribed cutoffs. Data showed that such is completely rejected and a more sensitive instrumentation should be preferred.

Investigation of the Small Size of Nanobodies for a Sensitive Fluorescence Polarization Immunoassay for Small Molecules: 3-Phenoxybenzoic Acid, an Exposure Biomarker of Pyrethroid Insecticides as a Model.

Limited reports on the use of nanobodies (Nbs) in fluorescence polarization immunoassay (FPIA) aroused us to explore if the small size of Nbs is a drawback for the development of sensitive FPIA to small molecular compounds, particularly since FPIA is a technology strongly dependent on molecular weight. In the present work, three different molecular weight Nbs against 3-phenoxybenzoic acid (3-PBA), an exposure biomarker of pyrethroid insecticides, including bare Nbs (15 kDa), Nbs-Avidin (Nbs-AV, 60 kDa), and Nbs-Alkaline phosphatase (Nbs-AP, 130 kDa) were specifically generated to cover distinct regions on the polarization and molecular weight relationship curve for a fluorescein tracer. In competitive FPIA, similar half-maximal inhibitory concentrations (IC50) of 3-PBA of 16.4, 12.2, and 14.8 ng mL-1 were obtained for Nbs, Nbs-AV, and Nbs-AP, respectively, indicating that the size of Nbs in the range tested had no significant effect on the sensitivity of the resulting competitive FPIA. An IC50 of 20.2 ng mL-1 for an anti-3-PBA polyconal antibody based FPIA further demonstrated the performance of Nbs, which was comparable to that of traditional antibodies in FPIA. Spike-recovery studies showed good and reproducible recovery of 3-PBA in urine samples, demonstrating the applicability of Nb-based FPIA. Overall, our results show that Nb-based FPIA achieves sensitivity levels of FPIA based on conventional antibodies and further indicate that Nb absolutely meets the sensitivity requirement of FPIA.

Development of a simple, rapid and high-throughput fluorescence polarization immunoassay for glycocholic acid in human urine.

In this paper, a simple, rapid and high-throughput fluorescence polarization immunoassay (FPIA) based on polyclonal antibodies (PAb) is described for the determination of glycocholic acid (GCA) in human urine. Three fluorescein-labeled GCA (tracers) with different structures and spacer bridges were synthesized and purified by thin-layer chromatography (TLC). The structure effect of tracers on the assay was investigated and the sensitivity of best tracer in the optimized FPIA demonstrated an IC50 value of 306 ng/mL. The working range of FPIA was 36 ∼ 2 600 ng/mL and the limit of detection (LOD) was 9 ng/mL. The developed FPIA was time-saving that could be completed within 10 min. Human urine samples spiked with GCA were analyzed by this method, followed by confirmation with commercial enzyme immunoassay analysis (EIA). Excellent recoveries and correlation between these two methods were observed (R2 = 0.996), suggesting the developed FPIA could be applied to screening of GCA in human urine samples without complicated cleanup.

Development of a Transcreener kinase assay for protein kinase A and demonstration of concordance of data with a filter-binding assay format.

A Transcreener kinase fluorescence polarization (FP) assay has been developed for the serine/threonine kinase protein kinase A (PKA). The PKA Transcreener kinase assay is an homogenous, competitive antibody-based FP assay that uses Far Red Alexa Fluor 633-labeled adenosine 5' disphosphate (ADP) tracer and mouse monoclonal anti-ADP antibody. The Transcreener PKA assay was validated with both known PKA inhibitors and library compounds. The Transcreener PKA assay is resistant to low-wavelength (or common) fluorescent interference from small-molecule library compounds and generates IC50 results comparable with current radioactive filter-binding assay.

Fluorescence Polarization Immunoassay Based on a New Monoclonal Antibody for the Detection of the Zearalenone Class of Mycotoxins in Maize.

To develop a sensitive fluorescence polarization immunoassay (FPIA) for screening the zearalenone class of mycotoxins in maize, two new monoclonal antibodies with uniform affinity to the zearalenone class and four fluorescein-labeled tracers were prepared. After careful selection of appropriate tracer-antibody pairs in terms of sensitivity and specificity, a FPIA that could simultaneously detect the zearalenone class with similar sensitivity was developed. Under optimum conditions, the half maximal inhibitory concentrations of the FPIA in buffer were 1.89, 1.97, 2.43, 1.99, 2.27, and 2.44 µg/L for zearalenone, α-zearalenol, ß-zearalenol, α-zearalanol, ß-zearalanol, and zearalanone, respectively. The limit of detection of FPIA for the zearalenone class was around 12 µg/kg in maize, and the recoveries ranged from 84.6 to 113.8%, with coefficients of variation below 15.3% in spiked samples. Finally, the FPIA was applied for screening naturally contaminated maize samples, and the results indicated a good correlation with that of high-performance liquid chromatography-tandem mass spectrometry.

Biocompatible, nanogold-particle fluorescence enhancer for fluorophore mediated, optical immunosensor.

Fluorophores have been used as effective signal mediators for detecting biomarkers in biosamples. The enhancement of the fluorescence can, therefore, improve the sensitivity of fluorophore-mediated biosensors. A nanogold particle (NGP), when placed at an appropriate distance from a fluorophore, can effectively enhance the fluorescence by transferring the free electrons of the fluorophore, normally used for self-quenching, to the strong surface plasmon polariton field (SPPF) of the NGP. We found that some organic solvents can also enhance the fluorescence significantly. To maximize the fluorescence enhancement, novel, biocompatible nanogold particle reagents (NGPRs) were developed by combining NGPs and biocompatible solvents and tested. The level of enhancement by NGPRs was found to be additively contributed by two enhancers. These NGPRs were able to increase the signal of a fiber-optic biosensor as much as 10 times and accurately quantify some of the important cardiac markers at a tens of picomolar level. These novel enhancers are expected to be effective for fluorophore-mediated bioimaging as well as biosensing.

Development of a non-radioactive, 384-well format assay to detect inhibitors of the mitogen-activated protein kinase kinase 4.

Mitogen-activated protein kinase (MAPK) kinases (MKKs, also called MAPK/extracellular signal-regulated kinase [ERK] kinase [MEK]) are constituents of numerous signal transduction pathways involved in growth, differentiation, and stress response. One of its members, MKK4, directly phosphorylates and activates the c-Jun terminal kinases (also called stress-activated protein kinase [SAPK]) in response to stress and pro-inflammatory cytokines. Recent evidence suggest that control of MKK4 activity may provide a novel approach for the treatment of cancer or as anti-inflammatory therapy. To screen for novel low-molecular-weight inhibitors of MKK4, we established a quantitative, non-radioactive in vitro kinase assay. Human MKK4 was expressed as fusion protein with glutathione S-transferase (GST) in Escherichia coli. Co-expression of a constitutive active fragment of the MAPK/ERK kinase kinase-1 yielded active GST-MKK4 using GST-SAPK alpha-kinase-negative (KN) mutant as substrate. We determined the kinetic constants for ATP and GST-SAPK alpha-KN. The apparent Km value for GST-SAPKalpha-KN was 3.7 microM, while the apparent Km value for ATP was 0.17 microM. Staurosporine inhibited GST-MKK4 with an IC50 of 70 nM. The kinase assay was adapted to a 384-well non-radioactive format. After the kinase reaction the phosphorylated product was captured onto a streptavidin-coated microtiter plate, and phosphorylation was detected with a europium-labeled anti-phosphotyrosine antibody, which allowed time-resolved fluorescence measurement.

A compact CMOS biochip immunosensor towards the detection of a single bacteria.

Recent use of biological warfare (BW) agents has led to a growing interest in the rapid and sensitive detection of pathogens. Therefore, the development of field-usable detection devices for sensitive and selective detection of BW agents is an important issue. In this work, we report a portable biochip system based on complementary metal oxide semiconductor (CMOS) technology that has great potential as a device for single-bacteria detection. The possibility of single-bacteria detection is reported using an immunoassay coupled to laser-induced fluorescence (LIF) detection. Bacillus globigii spores, which are a surrogate species for B. anthracis spores, were used as the test sample. Enzymatic amplification following immunocomplex formation allowed remarkably sensitive detection of B. globigii spores, and could preclude a complicated optical and instrumental system usually required for high-sensitive detection. Atomic force microscopy (AFM) was employed to investigate whether B. globigii spores detected in the portable biochip system exist in single-cell or multicellular form. It was found that B. globigii spores mostly exist in multicellular form with a small minority of single-cell form. The results showed that the portable biochip system has great potential as a device for single-particle or possibly even single-organism detection.

Development of fluorescence change-based, reagent-less optic immunosensor.

A reagent-less, regenerable and portable optic immunosensor was developed. A model sample, immunoglobulin G (IgG), was detected with this system based on changes in fluorescent intensity of fluorescent labeled protein A with specific reactivity to IgG depending on a reaction between the proteins. A glass plate immobilized with Qdot-labeled protein A was placed on the top of optic fibers designed for both excitation and fluorescence emission. The optic fibers with the Qdot-labeled protein A-immobilized glass plate were inserted into a solution of pH 7.4 phosphate buffered saline. After stabilization of the fluorescence intensity, IgG was added and the time-course of the fluorescence intensity was measured on a fluorometer connected with the optic fibers. Furthermore, the fluorescence response of a transient state was evaluated with the same system. When the Qdot-labeled protein A bound to IgG, fluorescence intensity decreased because of the inhibition by IgG. The degree of fluorescence decrease depends on the IgG concentration at a steady state and also in a transient state.

A highly sensitive and class-specific fluorescence polarisation assay for sulphonamides based on dihydropteroate synthase.

We describe a fluorescence polarisation assay based on the use of dihydropteroate synthase (DHPS) and a fluorescence probe for multi-sulphonamide detection. Dihydropteridine pyrophosphate (DHPPP) was synthesised and acts as the first substrate for DHPS. Under optimised conditions, the half-maximal inhibitory concentrations (IC50) of the assay were less than 100 ng mL(-1) for at least 29 sulphonamides and the time needed for the detection was less than 20 min. More importantly, the assay revealed quite uniform affinities for all of the individual sulphonamides tested, which has never before been achieved in an antibody-based assay.

[Comparative study of two techniques of ciclosporine monitoring]. / Étude comparative de deux techniques de dosage de la ciclosporine.

Ciclosporine (CsA) is an immunosuppressant drug used in bone marrow transplantation in order to extend allograft survival. Despite its efficiency, CsA can expose to therapeutic failure or to toxicity because of underdosing or overdosage. So, many techniques of monitoring CsA in blood were used, the referance one is the chromatographic technique then, the automated techniques: fluorescence polarization immunoassay (FPIA) and chimiluminescent microparticle immunoassay (CMIA). In this study, we aimed to compare the results of CsA concentrations measured by the two automised techniques. Statistical studies showed that the two techniques were repeatable and reproductible. Results obtained by FPIA were slightly higher than those obtained by CMIA but without a significative difference. In conclusion, FPIA technique could be used to measure CsA blood concentration in replacement of CMIA in case of technical problems.

High-sensitivity miniaturized immunoassays for tumor necrosis factor alpha using microfluidic systems.

We use microfluidic chips to detect the biologically important cytokine tumor necrosis factor alpha (TNF- alpha) with picomolar sensitivity using sub-microliter volumes of samples and reagents. The chips comprise a number of independent capillary systems (CSs), each of which is composed of a filling port, an appended microchannel, and a capillary pump. Each CS fills spontaneously by capillary forces and includes a self-regulating mechanism that prevents adventitious drainage of the microchannels. Thus, interactive control of the flow in each CS is easily achieved via collective control of the evaporation in all CSs by means of two Peltier elements that can independently heat and cool. Long incubation times are crucial for high sensitivity assays and can be conveniently obtained by adjusting the evaporation rate to have low flow rates of approximately 30 nL min(-1). The assay is a sandwich fluorescence immunoassay and takes place on the surface of a poly(dimethylsiloxane)(PDMS) slab placed across the microchannels. We precoat PDMS with capture antibodies (Abs), localize the capture of analyte molecules using a chip, then bind the captured analyte molecules with fluorescently-tagged detection Abs using a second chip. The assay results in a mosaic of fluorescence signals on the PDMS surface which are measured using a fluorescence scanner. We show that PDMS is a compatible material for high sensitivity fluorescence assays, provided that detection antibodies with long excitation wavelength fluorophores ( > or =580 nm) are employed. The chip design, long incubation times, proper choice of fluorophores, and optimization of the detection Ab concentration all combine to achieve high-sensitivity assays. This is exemplified by an experiment with 170 assay sites, occupying an area of approximately 0.6 mm(2) on PDMS to detect TNF-alpha in 600 nL of a dendritic cell (DC) culture medium with a sensitivity of approximately 20 pg mL(-1)(1.14 pM).

Stable, sensitive, fluorescence-based method for detecting cAMP.

cAMP is a universal secondary messenger that connects changes in the extracellular environment, as detected by cell surface receptors, to transcriptional changes in the nucleus. Since cAMP-mediated signal transduction plays a role in critical cell functions and human diseases, monitoring its activity can aid in understanding these responses and the process of drug discovery. This report examines the performance of a fluorescence-based competitive immunoassay in 384-well microplate format. Using purified cAMP as a competitor the estimated detection limit was determined to be 0.1 nM and Z'-factor was greater than 0.83, which indicates that the assay is of high quality and one of the most sensitive assays currently on the market. Of note, the results obtained were similar whether the reaction was allowed to proceed for 10 min or up to 60 min. Next, HEK 293 cells were treated with the promiscuous adenylate cyclase activator, forskolin, and the beta-adrenoceptor agonist, isoproterenol. The resultant average EC50 values were 11 microM and 123 nM, respectively, which correspond to those found in the literature. Together, these results demonstrate that this assay is afast, accurate, non-radioactive method that is ideal for high-throughput screening.

Stopped-flow fluorescence polarization immunoassay.

A general survey of the analytical application of kinetic methodology in fluorescence polarization immunoassay (FPIA) is presented. Stopped-flow mixing technique (SF) allows the initial rate of the immunochemical reaction between the tracer and the antibody to be obtained, which is used as the analytical parameter instead of the equilibrium signal used in conventional FPIA. The instrumentation required is described and the features of the analytical methods proposed are compared with those obtained by conventional FPIA. The usefulness of SF-FPIA for routine screening in clinical, environmental and food analysis is discussed.

A rapid detection method for Vaccinia virus, the surrogate for smallpox virus.

Prior to the World Health Organization's announcement of total eradication in 1977 [J. Am. Med. Assoc. 281 (1999) 1735], smallpox was a worldwide pathogen. Vaccinations were ceased in 1980 and now with a largely unprotected world population, smallpox is considered the ideal biowarfare agent [Antiviral Res. 57 (2002) 1]. Infection normally occurs after implantation of the virus on the oropharyngeal or respiratory mucosa [J. Am. Med. Assoc. 281 (1999) 2127]. Smallpox virus can be detected from the throats of exposed individuals prior to onset of illness and prior to the infectious stage of the illness. A rapid, sensitive real-time assay to detect Variola virus (smallpox) has been developed using the Vaccinia virus, a surrogate of smallpox, as a target. Cyanine 5 dye-labeled anti-Vaccinia antibody was used in a sandwich immunoassay to produce a fluorescent signal in the presence of the Vaccinia virus. The signal was detected using the Analyte 2000 biosensor (Research International, Monroe, WA). The Analyte 2000 uses a 635 nm laser diode to provide excitation light that is launched into a polystyrene optical waveguide. Fluorescent molecules within the evanescent wave are excited and a portion of their emission energy recouples into the waveguide. A photodiode quantifies the emission light at wavelengths between 670 and 710 nm. The biosensor was able to detect a minimum of 2.5 x 10(5) pfu/ml of Vaccinia virus in seeded throat culture swab specimens.

Fluorescence quenching competitive immunoassay in micro droplets.

A fluorescence quenching competitive immunoassay in micro droplets was applied to the sensitive detection of the pyrethroid insecticide, esfenvalerate. Laser induced fluorescence from rhodamine dye was used as a marker. The competitive immunoreaction was performed in micro droplets generated by a vibrating orifice aerosol generator system with a 10-microm diameter orifice. Fluorescence that was emitted from the droplets was detected by a 1/8 m imaging spectrograph with a 512 x 512 thermoelectrically cooled, charged-coupled device camera. The conjugate of esfenvalerate with rhodamine exhibited similar fluorescence to that of pure rhodamine 6G. When anti-esfenvalerate antibodies were added to the droplets, the fluorescence decreased. The reduction in emission was due to a strong quenching effect that arises from the interaction between the protein and rhodamine molecules following the antigen-antibody reaction. When a sample of esfenvalerate was added to the droplets, the release of the conjugated rhodamine from the antigen-antibody complex allowed the fluorescence signal to recover. An assay in a picoliter droplet sample was shown to enable detection down to approximately 0.1 nM. A very small mass of analyte could be detected with this method. A sample of river water was used to gauge the impact of matrix effects and was shown to give rise to negligible interference with the immunoassay.

Verification of performance with the automated direct optical TIRF immunosensor (River Analyser) in single and multi-analyte assays with real water samples.

In order to verify the reproducibility, precision, and robustness of the optical immunosensor River Analyser (RIANA), we investigated two common statistical methods to evaluate the limit of detection (LOD) and the limit of quantification (LOQ). Therefore, we performed a simultaneous multi-analyte calibration with atrazine, bisphenol A, and estrone in Milli-Q water. Using an automated biosensor, it was possible for the first time to achieve a LOD below 0.020 microg L(-1) using a common statistically based method without sample pre-treatment and pre-concentration for each of the analytes in a simultaneous multi-analyte calibration. This biosensor setup shows values comparable to those obtained by more classical analytical methods. Based on this calibration, we measured spiked and un-spiked real water samples with complex matrices (samples from different water bodies, from ground water sources, and tap water samples). The comparison between our River Analyser and common analytical methods (like GC-MS and HPLC-DAD) shows overall comparable values for all three analytes. Furthermore, a calibration of isoproturon (IPU) (in single analyte mode) resulted in a LOD of 0.016 microg L(-1), and a LOQ of 0.091 microg L(-1). In compliance with guidelines of the Association of Analytical Communities International (AOAC), six out of nine recovery rates (recovery rate: measured concentration divided by real concentration in percent) for three surface water samples with different matrices (spiked and un-spiked) could be obtained between 70 and 120% (recovery rates between 70 and 120%, as demanded by the guidelines of the AOAC International). The reproducibility was checked by measuring replica of each sample within independent repetitions. Robustness could be demonstrated by long-term stability tests of the biosensor surface. These studies show that the biosensor used offers the necessary reproducibility, precision, and robustness required for an analytical method.

Fabrication and characterization of 3D hydrogel microarrays to measure antigenicity and antibody functionality for biosensor applications.

We report the fabrication, characterization and evaluation of three-dimensional (3D) hydrogel thin films used to measure protein binding (antigenicity) and antibody functionality in a microarray format. Protein antigenicity was evaluated using the protein toxin, staphylococcal enterotoxin B (SEB), as a model on highly crosslinked hydrogel thin films of polyacrylamide and on two-dimensional (2D) glass surfaces. Covalent crosslinking conditions were optimized and quantified. Interrogation of the modified 3D hydrogel was measured both by direct coupling of a Cy5-labeled SEB molecule and Cy5-anti-SEB antibody binding to immobilized unlabeled SEB. Antibody functionality experiments were conducted using three chemically modified surfaces (highly crosslinked polyacrylamide hydrogels, commercially available hydrogels and 2D glass surfaces). Cy3-labeled anti-mouse IgG (capture antibody) was microarrayed onto the hydrogel surfaces and interrogated with the corresponding Cy5-labeled mouse IgG (antigen). Five different concentrations of Cy5-labeled mouse IgG were applied to each microarrayed surface and the fluorescence quantified by scanning laser confocal microscopy. Experimental results showed fluorescence intensities 3-10-fold higher for the 3D films compared to analogous 2D surfaces with attomole level sensitivity measured in direct capture immunoassays. However, 2D surfaces reported equal or greater sensitivity on a per-molecule basis. Reported also are the immobilization efficiencies, inter-and intra-slide variability and detection limits.

Highly sensitive biosensing using a supercritical angle fluorescence (SAF) instrument.

We present a new optical biosensor for probing molecular binding to a water/glass interface. The system is designed to measure the kinetics of surface reactions down to low analyte concentrations straightforwardly. The selective detection of surface bound fluorescence is achieved by collecting supercritical angle fluorescence (SAF) emission of surface bound molecules into the glass. Thereby the expansion of the detection volume into the aqueous probe is reduced to about one sixth of the fluorescence wavelength, consequently bulk fluorescence from the solution is rejected successfully. The SAF-signal is captured by a parabolic glass lens, which leads to high spatial collection efficiency and detection sensitivity. The sensor has an inverted optical design and is compatible with common glass cover slips, which strongly facilitates operation for the user working in the biological and biochemical fields. The performance of the system is demonstrated by real time measurements of antibody-antigen reactions. Rate constants of the reaction were extracted. Antigen concentrations were detected down to 10(-13) mol/l.