Simultaneous determination of human Enterovirus 71 and Coxsackievirus B3 by dual-color quantum dots and homogeneous immunoassay

Human Enterovirus 71 (EV71) and Coxsackievirus B3 (CVB3) have high risks for morbidity and mortality. A virus quantitation immunoassay has been proposed by employing two colored quantum dots (QDs), antibodies of the virus, and graphene oxide (GO). The QDs are streptavidin-conjugated quantum dots (SA-QDs), and the antibodies are biotinylated antibodies. Biotinylated EV71 antibody (Ab1) was associated with 525 nm green colored SA-QDs via biotin-streptavidin interaction forming QDs-Ab1, whereas biotinylated CVB3 antibody (Ab2) was associated with 605 nm red colored SA-QDs via biotin-streptavidin interaction forming QDs-Ab2. GO was an excellent quencher to the fluorescence of both QDs-Ab1 and QDs-Ab2. The targets of EV71 and CVB3 can break up the complex of QDs-Ab and GO, recovering the fluorescence of QDs-Ab1 and QDs-Ab2, respectively. Using these two different colored QDs-Ab fluorescence recovery intensities upon the addition of targets EV71 and CVB3, the two enteroviruses can be simultaneously quantitatively determined with a single excitation light. The detection limits of EV71 and CVB3 are 0.42 and 0.39 ng mL(-1) based on 3 times signal-to-noise ratio, respectively. More importantly, this strategy can be further used as a universal method for any protein or virus determination by changing the conjugated antibodies in disease early diagnosis, which can provide a fast and promising clinical approach for virus differentiation and determination. In a word, a simple, fast, sensitive, and highly selective assay for EV71 and CVB3 has been developed. It could be applied in clinical sample analysis with a satisfactory result. It was notable that the sensor could not only achieve rapid and precise quantitative determination of protein/virus by fluorescent intensity but also could be applied in semiquantitative protein/virus determination by digital visualization.     

Upconversion fluorescence resonance energy transfer in a homogeneous immunoassay for estradiol

We recently described a novel homogeneous assay principle based on upconversion fluorescence resonance energy transfer (UC-FRET), where an upconverting phosphor (UCP) is utilized as a donor. The UC-FRET has now been applied to a competitive homogeneous immunoassay for 17beta-estradiol (E2) in serum, using a small-molecular dye as an acceptor. The assay was constructed by employing an UCP coated with an E2-specific recombinant antibody Fab fragment as a donor and an E2-conjugated small-molecular dye, Oyster-556, as an acceptor. Standard curves for the assay were produced both in buffer and in male serum. Sensitized acceptor emission was measured at 600 nm under continuous laser diode excitation at 980 nm. In buffer, the IC50 value of the assay was 1 nM and in serum 3 nM. The lower limits of detection (mean of zero calibrators, 3 SD) were 0.4 and 0.9 nM, respectively. The measurable concentration range extended up to 3 nM in buffer and 9 nM in serum. Equilibrium in the assay was reached in 30 min. The novel principle of UC-FRET has unique advantages compared to present homogeneous luminescence-based methods and can enable an attractive assay system platform for clinical diagnostics and for high-throughput screening approaches.     

Sequential determination of two proteins by temperature-triggered homogeneous chemiluminescent immunoassay

A novel protocol for performing a sequential dual-protein immunoassay, based on a temperature-triggered separation/mixing process and HRP-catalyzed chemiluminescence (CL) detection, is described. In contrast to current multilabel-based detection techniques, a single HRP label is employed in this proposed method. Herein we introduce poly(N-isopropylacrylamide) (PNIP) and magnetic beads as bimolecular immobilizing carriers to separate different targets by taking advantage of thermal response, as demonstrated by sequential detection of human IgG and IgA. PNIP is known to aggregate and precipitate out of water when the temperature is raised above the lower critical solution temperature (LCST) of 31 degrees C; thus, it can be separated from supernatant by centrifugation. Besides, magnetic beads can be separated from PNIP by magnetic force as the temperature is lower than LCST. A homogeneous noncompetitive ELISA was employed, formed by primary antibodies immobilized onto the surface of magnetic beads and PNIP, antigen as IgG and IgA in the sample, and HRP-labeled second antibodies. Moreover, highly sensitive CL detection of HRP was applied, and the detection limits of IgG and IgA were as low as 2.0 and 1.5 ng/mL, respectively. Within the calibrated amount, the protocol had excellent precision within 11% for each target and was comparable in performance to commercial single-analyte ELISAs. Furthermore, the proposed method has been successfully applied to the determination of dual analyte in real samples without cross-reaction, and a good correlation was achieved after comparison with the conventional assay for IgG and IgA in 40 human serum samples.     

One-step homogeneous immunoassay for small analytes

We have developed a one-step, homogeneous noncompetitive immunoassay for small analytes using recombinant antibodies and morphine as the model analyte. A highly specific antibody against the immune complex (IC) formed between an anti-morphine antibody and morphine was selected from a naive scFv phage display library. The in vitro phage library selection procedure avoids the difficulties associated with the production of anti-IC antibodies by animal immunization. The anti-morphine and the anti-IC antibodies were labeled with a pair of fluorescence resonance energy transfer (FRET) fluorophores. In the FRET assay the labeled antibodies were incubated with saliva samples spiked with morphine, codeine, or heroin. Within 2 min, 5 ng/mL morphine, which is clearly under the recommended cutoff level, was detected without cross-reactivity to codeine or heroin. This assay principle is also widely applicable to other small analytes.     

A homogeneous and noncompetitive immunoassay based on the enhanced fluorescence resonance energy transfer by leucine zipper interaction

Fluorescence resonance energy transfer (FRET) between two GFP variants is a powerful technique to describe protein-protein interaction in a biological system. However, it has a limitation that the two variants tethered to the respective proteins have to be in sufficient proximity upon binding, which is often difficult to attain by simple N- or C-terminal fusions. Here we describe a novel method to significantly enhance FRET between GFP variant-tagged proteins with the use of leucine zippers. For the homogeneous sandwich immunoassay of a high molecular weight antigen human serum albumin (HSA), two separate single-chain Fvs recognizing distant epitopes of HSA were respectively fused with fluorescence donor ECFP or acceptor EYFP, and FRET between the two was analyzed by fluorescence spectrometry. Because these two proteins did not give any detectable FRET uponantigen addition, we tethered each protein with a leucine zipper motif (c-Jun or FosB) at the C-terminus to help the neighborhood of the GFP variants. Upon antigen addition, the new pairs showed significant antigen-dependent FRET. By exchanging the binding domains, the method will find a range of applications for the assay of other proteins and their interactions in vitro or in vivo.     

A homogeneous noncompetitive immunoassay for the detection of small haptens

We describe a noncompetitive homogeneous immunoassay for small haptens based on the antigen-dependent reassociation of antibody variable domains and beta-galactosidase (beta-gal) complementation (open sandwich enzymatic complementation immunoassay). As a model system, the reassociation of two fusion proteins, an anti 4-hydroxy-3-nitrophenylacetyl (NP) antibody heavy-chain variable-region fragment fused to an N-terminal deletion mutant of beta-gal (V(H)delta alpha) and the light-chain variable-region fragment fused to a C-terminal deletion mutant of beta-gal (V(L)delta omega), was monitored by the enzymatic complementation between the two. Upon simple mixing of the reagents with the sample, an antigen (NP)-dependent increase in enzymatic activity was observed. When 5-iodo-NP was measured, a 10 times higher sensitivity was observed, probably due to its higher affinity. Compared with our corresponding heterogeneous open sandwich enzyme-linked immunosorbent assay, approximately 1000-fold improvement in the sensitivity was attained, probably due to lower background V(H)-V(L) association. In addition, the assay required less time, handling, sample volume, and assay reagents.     

The inactivation of the non-enveloped enterovirus 71 (EV71) by a novel disinfectant gel formulation for topical use

Background: Hand-foot-mouth disease may cause severe central nervous system complications and even death, that is induced mainly by enterovirus 71 (EV71), which is a non-enveloped virus. Inactivation of the EV71 on hands could effectively inhibit the transmission. However, the inactivations of the EV71 by conventional disinfectants including the alcohols are poor, due to the high stability of the EV71. A novel pyridyl imidazolidinone compound (TJAB1099) was designed to specifically inhibit EV71 replication in vitro. It may potentially be developed as formulations applied on hands for EV71 transmission control.

Methods: The stress stability of TJAB1099 was first evaluated after storing in high temperature (60?°C, RH 10%), high humidity (25?°C, RH90%), and the high-intensity photolysis (4500?Lx ± 500?Lx) for 15 days, respectively. A wash-free antimicrobial gel containing the TJAB1099 was developed using the copolymer carrier. The antiviral activity, the acute oral toxicity, and the local irritation of the antimicrobial gel were evaluated accordingly.

Results: The results indicated that the TJAB1099 was stable during the storage in high temperature and humidity. However, a significant change (p?<?.0001) was detected when TJAB1099 stored in the high-intensity photolysis. The antimicrobial gel containing 1?μM TJAB1099 could inhibit EV71 significantly higher than the ethanol (75%) (p?<?.0001) and commercialized disinfectant products (p?<?.0001). The results of acute oral toxicity and the local irritation suggest that the TJAB1099 containing antimicrobial gel was not causing skin irritations and acute oral toxicity symptoms.

Conclusions: The results suggest that the antimicrobial gel containing TJAB1099 was safe and could effectively inhibit EV71 transmission in vitro.     

Long-wavelength fluorescence polarization immunoassay: determination of amikacin on solid surface and gliadins in solution

The versatility of the fluorescence polarization immunoassay (FPIA) is increased by using two long-wavelength labels, Nile Blue and a ruthenium(II) chelate. The first label has been used to study the potential of FPIA on a solid surface using dry reagent technology. The aminoglycoside antibiotic amikacin has been used as an analyte model, and the method has been applied to the analysis of serum samples. The second label has been used to show the practical application of FPIA to the determination of macromolecules, using gliadins as an analyte model, which have been determined in gluten-free food. Very low amounts of anti-amikacin antibodies and amikacin-Nile Blue tracer were immobilized onto nitrocellulose membranes, for the development of the amikacin method, and the consumption of reagents is lower than in conventional FPIA. Only the addition of the standard or sample extract at an adequate pH is required at the analysis time. The analyte displaces the tracer from the tracer-antibody immunocomplex, obtaining a decrease in the fluorescence polarization proportional to the analyte concentration. The gliadin-Ru(II) chelate tracer shows a relatively long lifetime, which allows the observation of differences in fluorescence polarization values between the tracer-antibody complex and the tracer alone. The dynamic range of the calibration graphs for both analytes is 0.5-10 microg mL-1 and the detection limits are 0.1 and 0.09 microg mL-1 for amikacin and gliadins, respectively. The study of the precision gave values of relative standard deviations lower than 5 and 1.5% for the amikacin and gliadin methods, respectively. Amikacin was determined in human serum samples using a previous deproteinization step with acetonitrile, obtaining recovery values in the range 83.4-122.8%. The gliadin method was applied to the analysis of gluten-free food samples by using a previous extraction step. The recovery study gave values between 94.3 and 105.0%.     

Development and validation of a one-step immunoassay for determination of cadmium in human serum.

A sensitive and simple one-step immunoassay was developed and validated for quantitative determination of Cd(II) in human serum. In this method, a monoclonal antibody that recognizes Cd(II)-EDTA complexes was directly immobilized onto microwell plates. The serum sample containing metallothionein(MT)-bound and non-MT-bound Cd(II) was acidified to displace the Cd(II) from MT. The sample was then treated with metal-free EDTA to convert Cd(II) to Cd(II)-EDTA complexes. A mixture of Cd(II)-EDTA complexes derived from serum samples and Cd(II)-EDTA conjugated with peroxidase enzyme was incubated in the wells to compete for binding sites of the immobilized antibody. After addition of peroxidase substrate, the bound fraction of the enzyme conjugate was measured by a microplate reader, and the signal was inversely proportional to the concentration of the Cd(II) in the sample. The assay limit of detection was 0.24 microg/L, and the effective working range at coefficient of variation of < or = 10% was 0.24-100 microg/L. Analytical recovery of spiked Cd(II), in the concentration range between 0.8 and 50 microg/L, was 97.8 +/- 4.0%. The assay was selective for Cd(II); other metal ions (Mn, Co, Cu, Zn, Mg, Hg, Ca, Ni, Fe, and Pb), tested at concentrations considerably higher than those present in human serum, did not significantly interfere with the assay. The assay results correlated well with those obtained by graphite furnace atomic absorption spectrometry (r = 0.984).     

Miniaturization of a homogeneous fluorescence immunoassay based on energy transfer using nanotiter plates as high-density sample carriers.

The miniaturization of a homogeneous competitive immunoassay to a final assay volume of 70 nL is described. As the sample carrier, disposable plastic nanotiter plates (NTP) with dimensions of 2 x 2 cm2 containing 25 x 25 wells, corresponding to approximately 15,000 wells on a traditional 96-well microtiter plate footprint, were used. Sample handling was accomplished by a piezoelectrically actuated micropipet. To reduce evaporation while pipetting the assays, the NTP was handled in a closed humid chamber and cooled to the point of condensation. To avoid washing steps, a homogeneous assay was developed that was based on energy-transfer (ET). As a model system, an antibody-based assay for the detection of the environmentally relevant compound, simazine, in drinking water was chosen. Antibodies were labeled with the long-wavelength-excitable sulfoindocyanine dye Cy5 (donor), and a tracer was synthesized by labeling BSA with a triazine derivative and the acceptor dye Cy5.5. At low analyte concentrations, the tracer was preferably bound to the antibody binding sites. As a result of the close proximity of Cy5.5 and Cy5, an efficient quenching of the Cy5 fluorescence occurred. Higher analyte concentrations led to a progressive binding of the analyte to the antibody binding sites. The increased Cy5 fluorescence was determined by using a scanning laser-induced fluorescence detector. The limit of detection (LOD), using an antibody concentration of 20 nM, was 0.32 microg/L, or 1.11 x 10(-16) mol of simazine. In comparison, the LOD of the 96-well microtiter-plate-based ET immunoassay (micro-ETIA) was 0.15 microg/L, or 1.87 x 10(-13) mol. The LOD of the optimized micro-ETIA at 1 nM IgG, was 0.01 microg/L.     

Surface plasmon fluorescence immunoassay of free prostate-specific antigen in human plasma at the femtomolar level

This study describes the development of a surface plasmon fluorescence spectroscopy (SPFS)-based sandwich immunoassay, for the detection of free prostate-specific antigen (f-PSA). The commercial CM5 chip (Biacore) was integrated into the SPFS-based assay making use of its three-dimensionally extended dextran architecture, which offers a large protein retention capacity and alleviates the metal-induced fluorescence quenching. The performance of the f-PSA assay was investigated in buffer and in human plasma, respectively. In the human plasma, the limit of detection of f-PSA was estimated to be approximately 80 fM for 40 min of contact time, which adequately meets clinical requirements.     

Competitive quenching fluorescence immunoassay for chlorophenols based on laser-induced fluorescence detection in microdroplets

An improved biomonitoring system for the analysis of 2,4,6-trichlorophenol (TCP) in urine samples has been developed. The principle of the biosensor device is the detection of laser-induced fluorescence (LIF) in single microdroplets by a homogeneous quenching fluorescence immunoassay (QFIA). The competitive immunoassay occurs in microdroplets (d = 58,4 microm) produced by a piezoelectric generator system with 10-microm-diameter orifice. A continuous Ar ion laser (488 nm) excites the fluorescent tracer; its fluorescence is detected by a spectrometer attached to a 512 x 512 cooled, charge-coupled device camera. Fluorescence is quenched by specific binding of TCP polyclonal antibodies to the fluorescent tracer (hapten A-fluorescein); the quenching effect is diminished by the presence of the analyte. Thus, an increase in the signal is produced in a positive dose-dependent manner when TCP is present in the sample. In 10 mM PBS buffer, the IC50 of the LIF-microdroplet QFIA is 0.45 microg L(-1) reaching a LOD of 0.04 microg L(-1). The QFIA with the same reagents performed in microtiter plate format achieved a LOD of 0.36 microg L(-1) in buffer solution. Performance in human urine was similar to that observed in the buffer. A LOD of 1.6 ,g L(-1), with a dynamic range between 4 and 149.5 microg L(-1) in urine, was obtained without any sample treatment other than dilution with the assay buffer. The detectability achieved is sufficient for occupational exposure risk assessment.     

Determination of the magneto-optical relaxation of magnetic nanoparticles as a homogeneous immunoassay

The interaction between human eotaxin (hEotaxin) and its polyclonal antibody anti-human eotaxin (anti-hEotaxin) was investigated by means of a novel liquid-phase immunoassay using the magneto-optical relaxation of ferrofluids. The binding quality as well as kinetic properties of the binding partners was determined using specifically binding magnetic probes. For this purpose, magnetic nanoparticles (MNP; DDM128N, Meito Sangyo, Japan) were initially functionalized with streptavidin. The biotin-nylated antibody was conjugated with streptavidin-MNP applying the streptavidin-biotin binding system. Binding reactions were detected by measuring the relaxation of the optical birefringence signal occurring when a pulsed magnetic field is applied to the ferrofluid. The addition of hEotaxin to anti-hEotaxin conjugated MNP in different amounts yielded an enlargement of the mean relaxation time due to the formation of MNP aggregates. In order to express the observed increase of the particles' effective diameter in terms of elementary kinetic processes between antigen and antibody, a kinetic model was introduced. Here, the binding reactions are described by a process of stepwise polymerization. The obtained results were compared with data received from surface plasmon resonance biosensor analysis, a standard tool for biomolecular interaction analysis.     

Competitive immunoassay for microliter protein samples with magnetic beads and near-infrared fluorescence detection

A competitive immunoassay with near-infrared (NIR) fluorescence detection to analyze microliter biological samples with an amol limit of detection (LOD) is described. An important feature about this technique is that the immunoreaction and fluorescence detection are separated into two distinct steps, allowing for independent optimization. In the immunoreaction step, NIR fluorescence-labeled antigen (Ag) competes with the unlabeled analyte (Ag) for antibodies (Ab) immobilized on the surface of paramagnetic beads. A magnet is then used to separate the bound antigen from the free in the supernatant. As the amount of Ag in the sample increases, there is less binding between Ag and immobilized Ab; therefore, the amount of Ag in the supernatant is proportionally related to the amount of Ag in the sample. In the fluorescence detection step, aliquots of the supernatant are concentrated onto a protein binding membrane by a capillary blotting technique with an optimized 33 nL/min flow rate. The fluorescence of the blotted spots is detected with a NIR sensitive photon counting system that is optimized to an instrumental LOD of 30 000 fluorophore molecules. This competitive assay demonstrates a sample LOD of 400 pg/mL of unlabeled rabbit immunoglobulin G spiked into bovine serum. This design features low sample volumes and reagent consumption.     

Genetically engineered elastin-protein A fusion as a universal platform for homogeneous, phase-separation immunoassay

A simple and universal platform for competitive phase-separation immunoassay is reported based on a fusion protein composed of a temperature-responsive elastin-like polypeptide (ELP) and the antibody-binding staphylococcal protein A (SpA). The basic principle is to take advantage of the ability of SpA to bind a variety of antibodies with high affinity, allowing simple separation of antigen-antibody complex by thermal precipitation. The resulting ELP-SpA fusion was shown to preserve the ability to reversibly precipitate as well as its high affinity toward different IgGs and IgMs. As a model system, a competitive phase-separation immunoassay based on the ELP-SpA format was established for paclitaxel (taxol) with IC(50) (20.18 nM) and the lower detection limit (2.94 nM) very similar to those reported for the ELISA format. Unlike the heterogeneous interaction in ELISA, which decreases the antibody-binding activity, the reported homogeneous immunoassay not only alleviates this problem but also enables the potential for high-throughput automation. We believe that the reported ELP-SpA fusion will find applications not only as a powerful diagnostic tool for diverse analytes but also a potential useful tool for purification and immobilization of antibody.     

Instantaneous derivatization technology for simultaneous and homogeneous determination of multiple DNA targets

There are potential advantages, in terms of simplicity and speed, for detecting DNA hybridization steps directly without using any external labels, especially for the multiplexed assays. In the current paper, we describe the use of a carrier-resolved label-free multiplexed assay for the simultaneous detection of multiple DNA targets. Herein we demonstrate that this protocol, using three homogeneous carriers thermosensitive poly(N-isopropylacrylamide), polystyrene beads, and magnetic beads, respectively, for simultaneous determination of three short DNA fragments specific to hepatitis B virus. Briefly, one hybridization occurs between a mixture of three different capture probe DNAs immobilized onto three carriers and three targets in a single vessel, and then chemiluminescence (CL) detection proceeds via an instantaneous derivatization reaction between the specific CL reagent 3,4,5-trimethoxylphenylglyoxal (TMPG) and the guanine nucleotide-rich regions within the target DNA. An excellent linearity is found within the range between 0.1 and 6.0 pmol with the lowest detection limit of 100 fmol. In contrast to current encoding strategies, every hybridization signal for the corresponding DNA target in our protocol is uniquely immobilized onto one carrier vehicle with a unique and intrinsic physical-chemical signature. Moreover, an instantaneous derivatization reaction is employed for the label-free determination of three targets in a single vessel. In addition, a simple CL setup is employed to read the carrier code instead of an expensive and complicated flow cytometer or imaging system commonly used for multiplexed assays. Further signal amplification is achieved by employing three amplified DNAs for second hybridization, which include a guanine nucleobase-rich sequence domain for the generation of light and an additional tethered nucleic acid domain complementary with one of the target DNA as an amplification platform. Such simple amplified CL transduction allows detection of DNA targets down to the 15-fmol level. This new protocol also provided a good capability in discriminating perfectly complementary DNA from single-base mismatches and noncomplementary sequences. Overall, the protocol described here may have value in a variety of clinical, environmental, and biodefense applications for which the accurate quantitative analysis of multiple DNA targets is desired.     

Homogeneous noncompetitive immunoassay for 17beta-estradiol based on fluorescence resonance energy transfer

We previously presented a homogeneous noncompetitive assay principle based on quenching of the fluorescence of europium(III) chelate. This assay principle has now been applied to the measurement of 17beta-estradiol (E2) using europium(III) chelate labeled estradiol specific antibody Fab fragment (Eu(III)-Fab) as a donor and E2 conjugated with nonfluorescent QSY21 dye as an acceptor. Fluorescence could be measured only from those Eu(III)-Fab that were bound to E2 of the sample, while the fluorescence of the Eu(III)-Fab that were not occupied by E2 were quenched by E2-QSY21 conjugates. The performance of the assay was tested both in buffer and in the presence of serum. Because approximately half of the Fabs were incapable of binding to E2, the maximum quenching efficiency was only 55%. The lowest limits of detection were 18 and 64 pM in buffer and serum-based calibrators, respectively. The highest measurable concentrations were 0.4 nM in buffer and 1 nM in serum. The presented noncompetitive assay principle requires only one binder, and it can be applied to other haptens as well, providing that a suitable antibody is available.     

Label-free electrochemical immunoassay for the detection of human chorionic gonadotropin hormone

Here we report on a new and rapid immunoassay for the label-free voltammetric detection of human chorionic gonadotropin hormone (hCG) in urine. Monitoring the changes in the current signals of antibodies (Abs) before and after the binding of the antigen (Ag) provides the basis for an immunoassay that is simple, rapid, and cost-effective. Since hCG is found at highly elevated levels in pregnant female urine with the range of 30,000-200,000 mIU/mL (approximately 30-200 nM) by 8-10 weeks into pregnancy, its label-free electrochemical detection was achieved by using our method. The coverage of the electrode surface with the Ab and the incubation time with the target Ag were optimized for the detection of hCG. The limit of detection of our method was calculated to be 15 pM (n = 3, approximately 15 mIU/mL) in synthetic hCG samples and 20 pM (n = 3, approximately 20 mIU/mL) in human urine. The electrochemical results for the detection of hCG in the urine samples were in agreement with the results obtained using a reference system, enzyme-linked immunosorbent assay. Further research about the intrinsic electroactivity of Abs and their target molecules would surely provide new and sensitive screening assays, as well as extensive data regarding their interaction mechanisms.     

Experimental determination of the boundary condition for diffuse photons in a homogeneous turbid medium

We present a simple experimental method that permits an empirical determination of the effective boundary condition and the extrapolated end point for the diffuse photon density in a homogeneous turbid medium.