Upconversion fluorescence enables homogeneous immunoassay in whole blood.
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Blood Chemical Analysis↗
Biomedical subjects
Timo Lövgren
Publications and source records attributed to Timo Lövgren.
Sensitive Listeria spp. immunoassay based on europium(III) nanoparticulate labels using time-resolved fluorescence.
Listeria spp. are Gram-positive rod shaped bacteria found universally in the environment. Pathogenic Listeria monocytogenes is seldom harmful to healthy adults, but can cause serious disease, listeriosis, especially to pregnant women, neonates, and elderly or immunocompromised people. Conventional methods for screening Listeria in food samples are time consuming and laborious, involving the use of a range of liquid media and plate cultures. In the current study, the total analysis time was shortened by employing a sensitive Listeria assay, which was able to detect the bacteria in low concentrations. Sensitivity of the sandwich immunoassay was substantially improved by utilizing europium(III)-chelate containing latex nanoparticles as tracers. Each 107 nm nanoparticle contained approximately 31000 europium(III)-chelates which enhanced the specific activity of the label. The sensitive nanoparticulate immunoassay developed for Listeria spp. was performed in one-step and two-step formats. One-step assay was notably faster, 15 min, and simpler to execute having analytical sensitivity of 300 CFU/ml and a dynamic range of three orders of magnitude. The sensitivity, 20 CFU/ml, of the 4 h two-step assay clearly exceeded that of the one-step assay, and the dynamic range was nearly five orders of magnitude. Food and environmental samples were measured against a commercial L. monocytogenes immunoassay with good correlation. The developed sensitive assay enabled shorter sample enrichment times and, therefore, faster analysis of Listeria spp. Obviously the detection of several other bacteria can also be enhanced by applying the nanoparticle assay technology.
Colony Count, Microbial↗
Unfolding of the immunoglobulin light and heavy chains is required for the enzymatic removal of N-terminal pyroglutamyl residues.
To enable Edman sequencing of pyroglutamylated immunoglobulins, enzymatic deblocking by pyroglutamate aminopeptidase is performed, often with variable yield and compromised solubility. Recently, enzymatic deblocking of immunoglobulins without denaturation was described. Although the conditions ensured efficient removal of pyroglutamyl residues, we conclude that deblocking is preceded by denaturation, which results in aggregation of the immunoglobulins. To study the effect of folding status on deblocking we developed a methanol based deblocking solution, which preserved the enzymatic activity of pyroglutamate aminopeptidase, provided conditions compatible with sequencing and enhanced deblocking of electroblotted samples, as well. At 50 degrees C and 35% (v/v) methanol the immunoglobulin chains were completely aggregated, but the degree of deblocking was comparable to that obtained with the previously described method. At 37 degrees C, the immunoglobulins were partly aggregated, but the deblocked chains were completely in the insoluble fractions, whereas the soluble fractions had retained pyroglutamylation in both chains, suggesting that unfolding of the immunoglobulins is required for the excision of the pyroglutamates. Inspection of the structures of pyroglutamylated immunoglobulin and pyroglutamate aminopeptidase P. furiosus indicates that the enzyme requires the substrate in an extended conformation, a criterium, which we conclude not to be fulfilled in the native form of immunoglobulins. Unfolding of the N-terminus would disrupt the immunoglobulin fold by breaking interactions between secondary structure elements and expose surfaces prone to aggregation.
Electrophoresis, Polyacrylamide Gel↗
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.
Estradiol↗
Homogeneous assay technology based on upconverting phosphors.
Upconversion photoluminescence can eliminate problems associated with autofluorescence and scattered excitation light in homogeneous luminescence-based assays without need for temporal resolution. We have demonstrated a luminescence resonance energy-transfer-based assay utilizing inorganic upconverting (UPC) lanthanide phosphor as a donor and fluorescent protein as an acceptor. UPC phosphors are excited at near-infrared and they have narrow-banded anti-Stokes emission at visible wavelengths enabling measurement of the proximity-dependent sensitized emission with minimal background. The acceptor alone does not generate any direct emission at shorter wavelengths under near-infrared excitation. A competitive model assay for biotin was constructed using streptavidin-conjugated Er3+,Yb3+-doped UPC phosphor as a donor and biotinylated phycobiliprotein as an acceptor. UPC phosphor was excited at near-infrared (980 nm) and sensitized acceptor emission was measured at red wavelength (600 nm) by using a microtitration plate fluorometer equipped with an infrared laser diode and suitable excitation and emission filters. Lower limit of detection was in the subnanomolar concentration range. Compared to time-resolved fluorometry, the developed assay technology enabled simplified instrumentation. Excitation at near-infrared and emission at red wavelengths render the technology also suitable to analysis of strongly colored and fluorescent samples, which are often of concern in clinical immunoassays and in high-throughput screening.
Biosensing Techniques↗
Locked nucleic acid (LNA) probes in high-throughput genetic analysis: application to an assay for type 1 diabetes-related HLA-DQB1 alleles.
OBJECTIVES: In large-scale genetic screening, an assay that is reliable, fast and easy to perform, and straightforwardly adapted to new analytes is a necessity. We describe a one-step assay for analyzing HLA-DQB1 alleles which are associated with susceptibility to type 1 diabetes. DESIGN AND METHODS: The assay is based on asymmetric PCR amplification and a homogeneous hybridization method. The specificity of the probes was improved by substituting LNA (locked nucleic acid) for DNA at the critical bases. RESULTS: The functionality of the LNA containing probes was found to be superior compared to probes consisting of DNA only. The homogeneous assay gave a correct genotyping result in 100% of the cases, which included both extracted DNA samples and blood samples dried on sample collection cards. CONCLUSION: This homogeneous approach provides a simple method to define disease risk associated with HLA alleles for large-scale screening projects.
Base Sequence↗
Simultaneous use of time-resolved fluorescence and anti-stokes photoluminescence in a bioaffinity assay.
A bioaffinity assay is described where anti-Stokes photoluminescence of inorganic lanthanide phosphors and time-resolved fluorescence of lanthanide chelates are measured from a single microtitration well without any disturbance from these label technologies to each other. Up-converting phosphor (UPC-phosphor) bioconjugate was produced by grinding the commercial, micrometer-sized UPC-phosphors to colloidal, submicrometer-sized phosphor particles and by attaching these phosphors to biomolecules. Experiments were carried out in standard 96-well microtitration plates to determine detection limits, linearity, and cross-talk of UPC-phosphor and europium chelate. In numbers of molecules the lower limits of detection for UPC-phosphor were roughly 3 x 10(3) particles in solution and 1 x 10(4) particles in solid phase, and for europium label same values were 9 x 10(6) and 9 x 10(7) molecules. Linearity of detection was for UPC-phosphor 5 orders of magnitude in solution and over 4 orders of magnitude in solid phase and for europium label over 5 orders of magnitude in solution and over 4 orders of magnitude in solid phase. The cross-talk between the two labels was practically nonexistent. In this study we show that up-converting anti-Stokes photoluminescent phosphors could be employed in bioaffinity assays as very potential labels with significant advantages either alone or together with long-lifetime lanthanide chelates.
Biological Assay↗
Synthesis, characterization, and application of Eu(III), Tb(III), Sm(III), and Dy(III) lanthanide chelate nanoparticle labels.
Preparation and characterization of europium(III), terbium(III), samarium(III), and dysprosium(III) polystyrene nanoparticle labels with lanthanide-specific fluorescence properties has been presented. Emulsion copolymerization of styrene and acrylic acid was used to synthesize uniform-sized nanoparticles approximately 45 nm in diameter. Europium(III) and samarium(III) lanthanides were chelated with 2-naphthoyltrifluoroacetone and trioctylphosphine oxide to dye the spherical particles, whereas terbium(III) and dysprosium(III) chelate complexes contained a newly synthesized ligand, 4-(2,4,6-tridecyloxyphenyl)pyridine-2,6-dicarboxylic acid. The fluorescence properties of the four lanthanides-including a wide Stokes shift, a narrow emission peak, and long fluorescence lifetime-were retained despite the incorporation into the nanoparticles. Furthermore, the nanoparticles, containing more than 1000 lanthanide chelates, were detectable at label concentrations 3 orders of magnitude lower than the corresponding soluble lanthanide chelate labels. The applicability of the labels prepared was demonstrated by a heterogeneous sandwich-type immunoassay for human prostate-specific antigen, where the lowest limits of detection of 1.6, 2.4, 10.1, and 114.2 ng/L were achieved using europium(III), terbium(III), samarium(III), and dysprosium(III) nanoparticles, respectively. The spectral and functional properties of the lanthanide-embedded polystyrene nanoparticles developed here suggest that the technology is applicable for high-sensitivity multicolor assays.
Chelating Agents↗
Photochemical characterization of up-converting inorganic lanthanide phosphors as potential labels.
We have characterized commercially available up-converting inorganic lanthanide phosphors for their rare earth composition and photoluminescence properties under infrared laser diode excitation. These up-converting phosphors, in contrast to proprietary materials reported earlier, are readily available to be utilized as particulate reporters in various ligand binding assays after grinding to submicron particle size. The laser power density required at 980 nm to generate anti-Stokes photoluminescence from these particulate reporters is significantly lower than required for two-photon excitation. The narrow photoluminescence emission bands at 520-550 nm and at 650-670 nm are at shorter wavelengths and thus totally discriminated from autofluorescence and scattered excitation light even without temporal resolution. Transparent solution of colloidal bead-milled up-converting phosphor nanoparticles provides intense green emission visible to the human eye under illumination by an infrared laser pointer. In this article, we show that the unique photoluminescence properties of the up-converting phosphors and the inexpensive measurement configuration, which is adequate for their sensitive detection, render the up-conversion an attractive alternative to the ultraviolet-excited time-resolved fluorescence of down-converting lanthanide compounds widely employed in biomedical research and diagnostics.
Immunoassay↗
Rapid and sensitive HBsAg immunoassay based on fluorescent nanoparticle labels and time-resolved detection.
The detection of hepatitis B virus in blood specimens is carried out commonly by measuring surface antigen (HBsAg) levels with assays designed for various random access immunoanalysers or rapid near-patient testing. These methods leave much to be desired in performance or throughput. The aim of this study was to develop a nanoparticle label-based rapid and sensitive HBsAg immunoassay and evaluate its performance compared to a well-established reference immunoassay (Enzygnost HBsAg 5.0). The assay developed is based on kinetic format and relies on one-step two-site antibody-antigen interaction. Europium(III)-chelate-doped nanoparticles and microtiter wells were coated with anti-HBsAg monoclonal antibodies specific for discrete epitopes. The adaptation of nanoparticle labels for quantitative HBsAg detection showed improved sensitivity (LLD: 0.028 ng/ml) and dynamics (up to 1000 ng/ml) with reasonably low coefficients of variation (concentration-CV%s 2.8-21.9%). Furthermore, concurrent sample runs with the ELISA reference method showed 100% agreement. The time required for the assay was only 10 min facilitating a rapid and convenient method for hepatitis B screening.
Europium↗
Enzyme inhibitor screening using a homogeneous proximity-based immunoassay for estradiol.
The authors have previously reported a homogeneous time-resolved fluorescence proximity immunoassay for estradiol. The assay was based on luminescence resonance energy transfer between a long lifetime fluorescent europium(III) chelate-dyed nanoparticle donor and a short lifetime, near-infrared fluorescent acceptor. The energy transfer prolonged the lifetime of the sensitized acceptor emission, and the fluorescence of the acceptor was measured using a time-resolved detection. The developed immunoassay was employed to screen inhibitors for enzyme 17beta-hydroxysteroid dehydrogenase type 1. The enzyme overexpressed in MCF-7 cells catalyzed a reversible conversion of estroneto17beta-estradiol. The inhibition efficiency of the tested molecule was obtained by comparing the final concentration of converted estradiol after 60 min of conversion reaction in a sample and in a conversion control not containing an inhibitor. The Zbeta factor calculated using the E2 concentrations of the homogeneous assay was 0.64, demonstrating a relatively good performance of the assay. The results from the homogeneous assay were comparable with the results obtained using radioactively labeled estrone as a substrate and high-performance liquid chromatography (HPLC) separation of estrone and converted estradiol after the enzyme reaction. Thus, this homogeneous assay can simplify the primary screening of potential new drug molecules by replacing a tedious radiometric HPLC method.
17-Hydroxysteroid Dehydrogenases↗
A sensitive adenovirus immunoassay as a model for using nanoparticle label technology in virus diagnostics.
BACKGROUND: Currently, PCR-based hybridization assays are widely applied in adenovirus diagnostics. However, the technology requires tedious sample preparation, and the amplification phase is susceptible to various contaminants leading to inconvenient and time-consuming assay procedure. Methods relying on viral antigen detection, e.g. immunofluorometric assays (IFMAs) and enzyme immunoassays (EIAs), are less complicated to carry out, but they provide limited sensitivity. OBJECTIVE: Our aim was to develop a simple and sensitive adenovirus assay based on direct antigen detection via sandwich-forming immunoreaction. The assay employed highly fluorescent europium(III)-chelate-doped nanoparticle labels and selection of high affinity monoclonal antibodies (anti-hexon) coated on label particles and microtitration wells. RESULTS AND CONCLUSIONS: The extremely high specific activity of the nanoparticle labels enabled the detection limit over 5000 virus particles per millilitre with purified virus particles. The sensitivity was improved by three orders of magnitude (800-fold) compared to concurrent time-resolved IFMA. Furthermore, the nanoparticle assay showed reasonably low coefficients of variation (4.0-20%) and excellent linearity of more than four orders of magnitude (from below 10(5) to 10(9) virus particles per millilitre). Analyzed nasopharyngeal patient specimens revealed a minor disturbance of matrix components, which could be avoided by dilution. The average signal difference between negative and positive samples was nearly four orders of magnitude. The developed assay was sensitive and more convenient approach to adenovirus screening compared to available assays. In addition, the study demonstrates the potential of nanoparticles in sensitive screening of viral analytes.
Adenovirus Infections, Human↗
Quantification of streptavidin adsorption in microtitration wells.
Streptavidin-coated microtitration plates have an important role as a solid phase in clinical diagnostics. We have designed techniques for evaluating quantitative and functional aspects of streptavidin adsorbed in microtitration wells. The theoretical monolayer adsorption capacity was modeled based on the molecular dimensions of the protein. Adsorbed streptavidin was quantified by direct labeling of protein with terbium chelate and with a sensitive bicinchoninic acid-based protein assay. A new small molecular weight (1037Da) reporter molecule, a europium-labeled biotin (Eu-biotin), was synthesized and used for monitoring adsorption and for determination of biotin-binding capacities of the streptavidin-coated wells. The theoretical monolayer adsorption of streptavidin yielded 6.20 pmol/cm(2) (370 ng) and consequently the theoretical adsorption capacity of a C12-format microtitration well (200 microl liquid, coated area 1.54 cm(2)) was 9.55 pmol/well (570 ng). Adsorption properties of streptavidin from two suppliers were tested, one of which yielded 350-380 ng/well while the other yielded over 500 ng/well. The biotin binding capacities were about 11 and 14 pmol/well, respectively. We managed to quantify surface-adsorbed streptavidin with sensitive fluorescence and protein measurement methods in the microtitration well. The new Eu-biotin reporter molecule enabled an exact and convenient determination of the biotin-binding capacities of streptavidin surfaces.
Adsorption↗
An all-in-one dry chemistry immunoassay for the screening of coccidiostat nicarbazin in poultry eggs and liver.
An automated immunoassay for the detection of nicarbazin residues in poultry eggs and liver was developed. The assay was based on a novel all-in-one dry chemistry concept and time-resolved fluorometry. The analyte specific antibody was immobilized into a single microtiter well and covered with an insulation layer, on top of which the label was dried in a small volume. The extracted sample was added automatically to the dry microtiter well, and the result was available within 18 min. Due to the rapidity and simplicity, the quantitative immunoassay could also be used as a high throughput screening method. The analytical limit of detection for the assay was calculated as 0.1 ng mL(-)(1) (n = 12) and the functional limit of detection as 3.2 ng g(-)(1) for egg (n = 6) and 11.3 ng g(-)(1) for liver (n = 6) samples. The sample recovery varied from 97.3 to 115.6%. Typically, the intra-assay variations were less than 10%, and interassay variations ranged between 8.1 and 13.6%.
Animals↗
Rapid time-resolved fluoroimmunoassay for the screening of narasin and salinomycin residues in poultry and eggs.
Anticoccidial drugs are extensively used in the poultry industry to control the infection of the single-cell protozoa of the genus Eimeria. The most commonly used coccidiostats in poultry are the polyether ionophores such as narasin and salinomycin. This paper presents a rapid and simple method for the screening of residues of these two coccidiostatic compounds in poultry and eggs. The method is based on time-resolved fluoroimmunoassay. Sample preparation of eggs consists only of one extraction and evaporation step, and a solid phase extraction step is needed only for the muscle sample preparation. Mean recoveries were 91.0% from muscle tissue and 81.1% from eggs for both narasin and salinomycin. The performance of the assay was evaluated only for narasin because salinomycin had a cross-reactivity of 100% in the assay, and the recoveries of the compounds were not significantly different (P >0.05). The limits of detection [mean + 3 x standard deviation (SD)] of narasin were 0.56 and 0.28 microg/kg, and the limits of quantification (mean + 9 x SD) were 1.80 and 0.57 microg/kg for muscle and eggs, respectively. The coefficients of variation (CV) of the interassay precision of the method, evaluated by five replicate analyses of muscle samples spiked with 2 microg/kg of narasin and egg samples spiked with 1 microg/kg of narasin, were 4.1 and 6.4%, respectively. The CVs of intra-assay precision tests, determined by 10 replicate analyses at the above-mentioned concentration levels, were 3.8 and 4.5%, respectively.
Animals↗
Immunoassay of total prostate-specific antigen using europium(III) nanoparticle labels and streptavidin-biotin technology.
Nanoparticle labels conjugated with biomolecules are used in a variety of different assay applications. We investigated the possibility of using europium(III)-labeled 68-nm nanoparticles coated with monoclonal antibodies or streptavidin (SA) to detect prostate-specific antigen (PSA) in serum. The selection of a suitable antibody pair and interference caused by the combination of nanoparticle label and structurally complex analyte were of special interest. A set of antibodies recognizing different epitope areas of PSA was mapped to find the optimal antibody pair for the immunometric nanoparticle-based assay. Different assay configurations were tested to obtain a good correlation with a conventional method based on biotinylated detection antibodies and europium(III) chelate-labeled streptavidin. Monoclonal capture antibody 5E4 was covalently coated on a microtitration well surface; biotinylated 5H6 monoclonal antibody (Mab) was used for detection, and europium(III)-labeled streptavidin-coated nanoparticles were utilized for signal generation. Total PSA concentrations were determined from a panel of male serum samples to test the developed assay. The correlation of the nanoparticle-based and reference assays was good; y=0.9844x-0.1252, R2=0.98, n=27; and the lowest limit of detection of the assay (LLD=0.83 ng/l) was 35-fold lower than for the reference method. The assay application presented here, where a structurally complex analyte is detected, combines the exceptionally high affinity of streptavidin-biotin technology and the high specific activity of long lifetime fluorescence nanoparticle labels. The general characteristics of this combination should permit the development of various immunoassay applications featuring high sensitivity, rapidity, and low consumption of reagents.
Antibodies, Monoclonal↗
Quantitative, rapid europium(III) nanoparticle-label-based all-in-one dry-reagent immunoassay for thyroid-stimulating hormone.
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Europium↗
Highly sensitive immunoassay of free prostate-specific antigen in serum using europium(III) nanoparticle label technology.
BACKGROUND: Recent proceedings in utilization of europium(III) chelate-dyed polystyrene nanoparticles as labels have combined the advantages of an enhanced monovalent binding affinity and a high specific activity of nanoparticle-antibody bioconjugate. Our objective was to evaluate the performance of the nanoparticle label technology with biological samples in an immunoassay of free prostate-specific antigen (PSA-F) using a standard microtitration well platform. METHODS: Long-lifetime luminescent europium(III)-chelate nanoparticles, 107 nm in diameter, were coated with a PSA-F specific monoclonal antibody. The two-step noncompetitive immunoassay was performed in a microtitration well coated with a second monoclonal antibody. The signal of the surface-bound nanoparticle-antibody bioconjugates was measured directly from the bottom of the well using a standard time-resolved plate fluorometer. RESULTS: The detection limit (mean + 2SD) of the nanoparticle-based PSA-F assay was 0.21 ng/l using a 20-microl sample volume. The assay response was linear up to 5 microg/l, and the functional sensitivity was approximately 0.5 ng/l. The within-run imprecision for spiked serum samples at concentrations 0.0005-0.5 microg/l was 6.4-21.8%, and the within-run and between-run imprecisions for serum samples at concentrations 0.2-2.5 microg/l were 3.4-7.2% and 4.4-7.6%, respectively. The concentrations obtained from serum samples correlated well with the reference immunoassay; slope = 1.018 +/- 0.018; intercept = 0.012 +/- 0.021 microg/l; S(y/x) = 0.112 microg/l; r = 0.993; n = 51. CONCLUSIONS: The developed method demonstrated acceptable performance characteristics allowing clinical studies utilizing patient samples with extremely low concentrations of PSA-F. The present assay detected PSA-F in most of samples from prostatectomized men and in few samples from healthy women that were nondetectable according to the reference immunoassay.
Europium↗