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.

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.

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.

[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.

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.

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.

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.

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.

Analytic performance evaluation of a new turbidimetric immunoassay for phenytoin on the ADVIA 1650 analyzer: effect of phenytoin metabolite and analogue.

Phenytoin is an anticonvulsant that requires therapeutic drug monitoring. Recently, Bayer HealthCare, Diagnostics Division released a turbidimetric immunoassay of phenytoin on the ADVIA 1650 analyzer. We evaluated the analytic performance of this assay by comparing values obtained in 52 patients receiving Phenytoin using this new assay with the values obtained by using a widely used fluorescence polarization immunoassay (FPIA). The new turbidimetric immunoassay for phenytoin showed the following imprecision with the low, medium, and high controls: total CV of 5.2% (mean 4.81 microg/mL), 3.7% (mean 16.24 microg/mL), and 4.1% (mean 22.65 microg/mL), respectively. The detection limit of the assay was 0.79 microg/mL, and the assay was linear up to a phenytoin concentration of 46.1 microg/mL. The assay showed excellent dilution recovery and recovery of spiked samples (mean recovery 101.4% and 94.4%, respectively). We observed an excellent correlation between the values obtained by the FPIA (x-axis) assay and the new turbidimetric (y-axis) assay (y=1.06 x-0.61, r=0.98, n=52). We also determined the cross-reactivity of 5-(p-hydroxyphenyl)-5-phenylhydantoin (HPPH), a major metabolite of phenytoin, and of oxaprozine, an analogue with a similar chemical structure to phenytoin, in both phenytoin assays. Both assays showed almost no cross-reactivity to oxaprozine and only small (5%-8%) cross-reactivity to HPPH. We also found that the turbidimetric assay was free from interference at least up to 1200 mg/dL of hemolysis, 30 mg/dL of free bilirubin, 34.5 mg/dL of conjugated bilirubin, and 750 mg/dL of triglyceride (Intralipid). When a drug-free serum was followed by a serum sample containing 38.5 microg/mL of phenytoin, no sample probe carryover effect was observed. We conclude that the new turbidimetric assay can be used for routine monitoring of phenytoin in clinical laboratories.

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 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.

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.

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).

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.

In situ measurement of degranulation as a biosensor based on RBL-2H3 mast cells.

A direct degranulation assay has been developed to enable the use of RBL mast cells as a biosensor for screening chemical libraries for drug discovery and environmental toxicity evaluation. Release of beta-hexosaminidase into the extracellular milleu is widely used to characterize cellular components and mechanisms involved in stimulated exocytosis, including those initiated by crosslinking of IgE receptors on mast cells. To adapt this versatile assay for high throughput screening, we developed a direct, in situ method in which beta-hexosaminidase detection is carried out in a single step, convenient for multi-sample processing and thus for biosensor applications. This direct assay is efficient for measuring exocytosis in antigen-stimulated RBL mast cells, detecting antigen concentrations as low as 1 pM. We also demonstrate its utility in detecting inhibition of degranulation by a known pharmacologic inhibitor that blocks Syk tyrosine kinase activity critical for cell activation.

Theoretical and experimental analysis of analyte transport in a fiber-optic, protein C immuno-biosensor.

Protein C (PC) is an important anticoagulant in human blood plasma, and early diagnosis of PC deficiency is critical for preventing dangerous thromboembolic complications. A fiber-optic PC immuno-biosensor has been under development in our research group for real-time PC-deficiency diagnosis. The sensor has demonstrated a good sensitivity and specificity for quantifying PC in buffered solutions. However, for plasma samples, with a limited sample reaction time, the sensor produced only 30% of the signal intensity of PC in buffer. The high plasma viscosity (1.9 cP) was speculated as the major reason for signal intensity reduction. In this investigation, the sensing performance of the fiber-optic PC biosensor is systematically characterized in terms of physical and chemical properties of the sample media. Theoretical and experimental analyses indicate that the reduced diffusion rate of PC molecules in viscous samples caused the sensing system to be more mass-transfer-limited. Convective flow of sample/reagent solutions during immunoreactions can increase the rate of the analyte mass transport from the bulk solution to the sensor surface, with reaction kinetics changing from mass-transfer-limited to reaction-limited as flow velocity increases. It was shown that PC sensor performance was significantly improved for plasma samples with convection. The effect of the flow velocity and incubation times for samples and reagents on the sensor performance was also systematically analyzed to optimize the assay protocol for PC sensing. Currently, a 6-cm-long immuno-biosensor is capable of quantifying PC in plasma (1 mL) in the heterozygous PC deficiency range (0.5 to 2.5 microg/mL) within 5 minutes, at an average signal-to-noise ratio of 50.

Comparison of serum digoxin concentration monitoring by fluorescence polarization immunoassay on the TDxFLx and dry chemistry enzyme immunoassay on the Vitros 950.

The aim of the study was to compare the results of digoxin assays performed using fluorescence polarization immunoassay (FPIA) on the TDxFLx and a dry chemistry enzyme immunoassay (EIA) on the Vitros 950. Within-run CV amounted to 8.52-10.49% for FPIA and 2.47-5.39% for EIA. Between-run CV amounted to 6.41-8.97% for FPIA and 3.40-5.04% for EIA. Analytical bias ranged from 2.57-4.0% for FPIA and from 9.86-11.9% for EIA. In comparative studies the correlation coefficient was 0.878; Deming regression analysis yielded a slope of 1.057 (95% CI: 0.573 to 1.541) and intercept of 0.078 (95% CI: -0.391 to 0.547), and the Passing-Bablok agreement test yielded a slope of 1.111 (95% CI: 0.988 to 1.212) and intercept of 0.094 (95% CI: -0.018 to 0.182). The mean digoxin concentration in patients' sera measured by EIA was significantly higher than that measured by FPIA (1.347 vs. 1.196 ng/ml, p<0.02). The mean absolute difference between results amounted to 0.146 ng/ml (95% CI: 0.0261 to 0.266). In comparison to EIA, FPIA yielded a higher number of subtherapeutic concentrations <0.5 ng/ml (29.7% vs. 21.8%) and a lower number of digoxin concentrations >1.2 ng/ml (25.7% vs. 35.6%). These discrepancies occurred in approximately 10% of samples. The obtained results showed different analytical performance and method-dependent differences in the distribution of results. This indicates the necessity to harmonize digoxin immunoassays if two different analytical systems are used in the same clinical setting.