Microbial identification from faces and urine in one step by two-photon excitation assay technique.

Two-photon excitation fluorometry (TPX) is a separation-free bioaffinity assay technique which enables accurate diagnostic testing in microvolumes. The technology is currently commercially applied in an automated mariPOC® test system for rapid phenotypic multi-microbe detection of pathogen antigens. The first TPX applications for diagnostics were intended for respiratory infection testing from nasopharyngeal and oropharyngeal samples. Feces and urine are more complex sample matrices and contain substances that may interfere with immunoassay binding or fluorescence detection. Our objective was to study the suitability of these complex matrices in the TPX technique. As expected, feces and urine elevated fluorescence levels but the methodology has the unique property of compensating for matrix effects. Compensation allows reliable separation of specific fluorescence from the fluorescence caused by the matrix. The studied clinical samples did not contain immunoassay inhibitors. The results suggest that the methodology is robust and may provide reliable testing of feces and urine samples with high accuracy.

A novel antibody avidity methodology for rapid point-of-care serological diagnosis.

A novel methodology is introduced for rapid serological diagnosis. This methodology combines the antibody bridging assay principle with the measurement of antibody avidity. The combination allows the determination of the infection phase with a single dilution of a single sample of serum. This is a significant improvement on current serological techniques which often require either paired-sample testing (IgG/IgM serology) or testing of the sample in several dilutions (IgG avidity testing). Assay methods were developed on two immunoassay platforms; the heterogeneous time-resolved fluoroimmunoassay and the separation-free two-photon excitation fluorometry. The new methods were compared to conventional class-specific IgG/IgM and IgG avidity techniques. The major findings were that the avidity results of the new methodology were independent of the sample dilution (specific antibody concentration in serum) and consistent between immunoassay platforms. This new methodology is simple, rapid, and quick to perform. It provides the possibility of running serodiagnostic tests at point-of-care with bench-top random-access analyzers.

Development of a rapid assay methodology for antimicrobial susceptibility testing of Staphylococcus aureus.

Development of a new phenotypic technique for rapid antimicrobial susceptibility testing (AST) of methicillin-resistant Staphylococcus aureus is presented. The new technique combines bacterial culturing and specific immunometric detection in a single separation-free process. The technique uses dry chemistry reagents and the recently developed two-photon excitation detection technology, which allows online detection of bacterium-specific growth. The performance of the new technique was evaluated by monitoring the growth of S. aureus reference strains and determining their susceptibility to oxacillin. In the direct analysis of clinical specimens, method specificity and tolerance to interferences caused by other bacteria present in the sample are pivotal. Other bacteria can compete with the bacteria of interest for nutrients, for example. Specificity and tolerance were studied against Staphylococcus epidermidis reference strains. The results suggest that the new technique could allow rapid AST directly from clinical samples within 6 to 8 h. Such a rapid and simple testing methodology would be a valuable tool in clinical microbiology because it would shorten the turnaround times of microbiologic analyses. Advantages of the new approach in relation to conventional methods are discussed.

Rapid method for detection of influenza a and B virus antigens by use of a two-photon excitation assay technique and dry-chemistry reagents.

New separation-free assay methods for the rapid detection of influenza A and B virus antigens are presented. The methods employ dry-chemistry reagents and the recently developed two-photon excitation (TPX) fluorescence detection technology. According to the assay scheme, virus antigens are sandwiched by capture antibody onto polymer microspheres and fluorescently labeled antibody conjugate. Consequently, fluorescent immunocomplexes are formed on the surface of microspheres in proportion to the concentration of the analyte in the sample. The fluorescence signal from individual microspheres is measured, separation free, by means of two-photon excited fluorescence detection. In order to demonstrate the applicability of the new assay technique for virus antigen detection, methods for influenza A and B viruses were constructed. The assay method for influenza A virus applied a molecular fluorescent label, whereas the method for influenza B virus required a nanoparticle fluorescent reporter to reach sufficient clinical sensitivity. The new methods utilize a dry-chemistry approach, where all assay-specific reagents are dispensed into assay wells already in the manufacturing process of the test kits. The performance of the assay methods was tested with nasopharyngeal specimens using a time-resolved fluoroimmunoassay as a reference method. The results suggest that the new technique enables the rapid detection of influenza virus antigens with sensitivity and specificity comparable to that of the reference method. The dose-response curves showed linear responses with slopes equal to unity and dynamic assay ranges of 3 orders of magnitude. Applicability of the novel TPX technique for rapid multianalyte testing of respiratory infections is discussed.

Quantitative detection of cell surface protein expression by time-resolved fluorimetry.

A method is introduced for quantitative detection of cell surface protein expression. The method is based on immunocytochemistry, the use of long decay time europium(III) chelate and platinum(II) porphyrin labels, and detection of photoluminescence emission from adhered cells by time-resolved fluorimetry. After immunocytochemistry, the assay wells are evaporated to dryness and measured in the dry state. This protocol allows repeated and postponed analysis and microscopy imaging. In order to investigate the performance of the method, we chose expression of intercellular adhesion molecule-1 (ICAM-1) of endothelial cell line EAhy926 as a research target. The expression of ICAM-1 on the cells was enhanced by introduction of a cytokine, tumour necrosis factor-alpha (TNFalpha). The method gave signal:background ratios (S:B) of 20 and 9 for europium and platinum labels, respectively, whereas prompt fluorescent FITC label gave a S:B of 3. Screening window coefficients (=Z'-factor) were >0.5 for all the three labels, thus indicating a score for an excellent screening assay. In conclusion, the method appears to be an appropriate choice for protein expression analysis, both in high-throughput screening applications, and for detailed sample investigation by fluorescent microscopy imaging.

Effect of polystyrene microsphere surface to fluorescence lifetime under two-photon excitation.

Molecular assays such as immunoassays are often performed using solid carriers and fluorescent labels. In such an assay format a question can be raised on how much the fluorescence of the label is influenced by the bio-affinity binding events and the solid carrier surface. Since changes in fluorescence intensity as labels bind to surfaces are notoriously difficult to quantify other approaches are preferred. A good indicator, independent of the fluorescence intensity of the label, is the fluorescence lifetime of the marker fluorophore. Changes in fluorescence lifetime reliably indicate the presence of dynamic quenching, energy transfer or other de-excitation processes. A microsphere based assay system is studied under two-photon excitation. Changes in fluorescence lifetime are studied as labeled protein conjugates bind on microsphere surfaces--both direct on the surface and with a few nanometer distance from the surface. Fluorescence signal is measured from individual polystyrene microspheres and the fluorescence lifetime histogram is simultaneously recorded. The results indicate that self-quenching and quenching by the polystyrene surface are both present in such a system. However, the effect of the surface can be avoided by increasing the distance between the surface and the label. Typical distances achieved by a standard sandwich type of assay, are already sufficient to overcome the surface induced quenching in fluorescence detection.

A novel separation-free assay technique for serum antibodies using antibody bridging assay principle and two-photon excitation fluorometry.

A new technique for separation-free detection of antigen-specific antibodies is presented. The new technique employs antibody bridging assay principle and the recently developed ArcDia TPX fluorescence detection technology. According to the assay scheme, antibody molecules from the sample bind with one arm to an antigen on polymer microspheres and with the other arm to a fluorescently labeled secondary antigen reagent. Consequently, fluorescent immunocomplexes are formed on the surface of microspheres in proportion to the concentration of the analyte in the sample. The fluorescence signal from individual microspheres is measured by means of two-photon excited fluorescence detection. In order to demonstrate the applicability of the new assay technique, an assay for anti-adenovirus antibodies was constructed. The function of the assay method was tested both with monoclonal anti-adenovirus antibody preparation (standard analyte), and with positive serum samples. Standard class-specific ELISA was used as a reference method. The new assay method provides comparable sensitivity and precision, and wider dynamic range for IgG antibodies than the ELISA method. The standard curve showed linear response (R(2)=0.999) with a dynamic range of three orders of magnitude, detection limit (mean+3S.D.) of 8 pM, and intra-assay signal precision of 5%. Applicability of the new method for clinical serodiagnostics is discussed.

A lab-on-a-chip compatible bioaffinity assay method for human alpha-fetoprotein.

A new lab-on-a-chip compatible binding assay platform is introduced. The platform combines dry-chemistry bioaffinity reagents and the recently introduced ArcDia TPX binding assay technique. The technique employs polymer microspheres as a solid phase reaction carrier, fluorescently labeled antibody conjugates, and detection of fluorescence emission from the surface of individual microspheres by two-photon excitation fluorescence. Signal response of the technique is independent of the reaction volume, thus the technique is particularly well suited for detection of bioaffinity reactions from miniature volumes. Performance of the new assay platform is studied by means of an immunometric assay of human alpha-fetoprotein (hAFP) in 384-plate format, and the results are compared to those of a corresponding wet-chemistry assay method. The results show that the ArcDia TPX detection technique can be combined with dry-chemistry reagents without compromises in assay performance. The microchip field has so far been characterized with a lack of microchip-compatible detection platforms which would allow cost-effective microchip design and sensitive bioaffinity detection. The presented detection technique is expected to provide a solution for this shortage.

Dipyrrylmetheneboron difluorides as labels in two-photon excited fluorometry. Part I--Immunometric assays.

Seven different two-photon excitable dipyrrylmetheneboron difluoride labels (dipyrrylmethene-BF(2) labels) and a frequently used TAMRA label were conjugated to mouse IgG against alpha-fetoprotein in variable substitution degrees. Altogether 40 IgG conjugates were prepared, and studied with respect to one-photon absorption and emission properties, and two-photon fluorescence efficiency using 1064 nm laser as illumination source. Performance of the IgG conjugates as tracers in a separation-free immunometric assay of alpha-fetoprotein was evaluated using two-photon excitation assay technology, ArcDia TPX. The results show that the dipyrrylmethene-BF(2) labels provide subpicomolar sensitivity, which is an order of magnitude better than that of TAMRA label. The effect of chromophore structure and substitution degree of IgG-label conjugates on the assay performance is discussed.

Dipyrrylmetheneboron difluorides as labels in two-photon excited fluorometry. Part II--Nucleic acid hybridization assays.

Five two-photon excitable dipyrrylmetheneboron difluoride labels (dipyrrylmethene-BF(2) labels) with fluorescence emission maximum between 530 and 590 nm, and a frequently used rhodamine label, TAMRA, were conjugated to aminomodified oligonucleotides. The performance of the labeled oligonucleotides was studied in a separation-free nucleic acid hybridization assay using ArcDia TPX bioaffinity assay technology. The results show that oligonucleotide conjugates of dipyrrylmethene-BF(2) labels provide higher two-photon excited fluorescence yield and better assay sensitivity than corresponding TAMRA conjugate. The effect of conjugation on photophysical properties of the labels and performance of the labeled oligonucleotides in separation-free hybridization assay is discussed.

Syntheses of novel dipyrrylmethene-BF2 dyes and their performance as labels in two-photon excited fluoroimmunoassay.

Two-photon excitation of fluorescence (TPE) has been found a powerful tool in the field of microscopy imaging and recently also in the field of bioanalytics. The recently introduced bioaffinity assay technology, ArcDia TPX, enables separation-free ultra-sensitive immunoassays from microvolumes. This assay technique is based on the use of microspheres as a solid reaction carriers and two-photon excited fluorescence detection. In the ArcDia TPX-technology, the individual microparticles are observed and the number of bound biomolecules on the microparticle surface is quantified by two-photon excited fluorescence. Here we present synthesis and use of a novel dipyrrylmethene-BF2 fluorophore that has been designed to be used as label in ArcDia TPX assay technique. The absorption and emission wavelengths of the label are tuned to allow excitation with a 1064 nm microchip laser. The label contains two-carboxylic residues, one of which is activated as N-hydroxysuccinimide ester to enable labeling of amino residues of biomolecules. The other carboxylic group is in free form to increase solubility in aqueous solutions. This new fluorescent label is tested in a separation-free immunoassay using ArcDia TPX assay technique. The performance of the new label is compared to that of one of the brightest fluorophores available, R-phycoerythrin (RPE). According to the results, the dipyrrylmethene-BF2 label provides significantly better signal-to-background ratio, leading to higher assay sensitivity and broader dynamic range compared to that of RPE. Good solubility to aqueous solutions and high fluorescence quantum efficiency, suggests the dipyrrylmethene-BF2 label is applicable also in other fluorescence-based applications.

Hydrophilic labeling reagents of dipyrrylmethene-BF2 dyes for two-photon excited fluorometry: syntheses and photophysical characterization.

Recently introduced bioaffinity assay technology, ArcDia TPX, is based on two-photon excited fluorescence (TPE) and it enables separation-free ultra-sensitive immunoassays from microvolumes. Here we present syntheses of novel two-photon excitable fluorescent labeling reagents which have been specially designed to be used as label molecules in the ArcDia TPX assay technique. The labeling reagents are based on dipyrrylmetheneboron difluoride (dipyrrylmethene-BF2) chromophore, which have been substituted with aryl, heteroaryl or arylalkenyl chemical groups to extend the pi-electron conjugation. These substitutions results in a series of dipyrrylmethene-BF2 fluorophores with different photophysical properties. Dipyrrylmethene-BF2 fluorophores have been further substituted with a dipeptide linker unit and finally activated as succinimidyl esters to enable specific coupling with primary amino groups. The dipeptide linker serves as a spacer arm between the label and a target, and enhances the solubility of the label in aqueous solutions. Study of the chemical and photophysical performance of the new labeling reagents is described. The new labeling reagents exhibit high fluorescence quantum yields, and molar absorption coefficients. The results show that the new labels with the hydrophilic dipeptide linker unit provide large two-photon excitation cross-sections, high fluorescence quantum efficiency and good solubility in aqueous solutions. The results suggest that the novel dipyrrylmethene-BF2 labels are highly applicable to bioaffinity assays based on two-photon excitation of fluorescence.

Efficient production of active chicken avidin using a bacterial signal peptide in Escherichia coli.

Chicken avidin is a highly popular tool with countless applications in the life sciences. In the present study, an efficient method for producing avidin protein in the periplasmic space of Escherichia coli in the active form is described. Avidin was produced by replacing the native signal sequence of the protein with a bacterial OmpA secretion signal. The yield after a single 2-iminobiotin-agarose affinity purification step was approx. 10 mg/l of virtually pure avidin. Purified avidin had 3.7 free biotin-binding sites per tetramer and showed the same biotin-binding affinity and thermal stability as egg-white avidin. Avidin crystallized under various conditions, which will enable X-ray crystallographic studies. Avidin produced in E. coli lacks the carbohydrate chains of chicken avidin and the absence of glycosylation should decrease the non-specific binding that avidin exhibits towards many materials [Rosebrough and Hartley (1996) J. Nucl. Med. 37, 1380-1384]. The present method provides a feasible and inexpensive alternative for the production of recombinant avidin, avidin mutants and avidin fusion proteins for novel avidin-biotin technology applications.

New separation-free assay technique for SNPs using two-photon excitation fluorometry.

A new separation-free method for detection of single nucleotide polymorphisms (SNPs) is described. The method is based on the single base extension principle, fluorescently labeled dideoxy nucleotides and two-photon fluorescence excitation technology, known as ArcDia trade mark TPX technology. In this assay technique, template-directed single base extension is carried out for primers which have been immobilized on polymer microparticles. Depending on the sequence of the template DNA, the primers are extended either with a labeled or with a non-labeled nucleotide. The genotype of the sample is determined on the basis of two-photon excited fluorescence of individual microparticles. The effect of various assay condition parameters on the performance of the assay method is studied. The performance of the new assay method is demonstrated by genotyping the SNPs of human individuals using double-stranded PCR amplicons as samples. The results show that the new SNP assay method provides sensitivity and reliability comparable to the state-of-the-art SNaPshot trade mark assay method. Applicability of the new method in routine laboratory use is discussed with respect to alternative assay techniques.

Fluorescent nanoparticles as labels for immunometric assay of C-reactive protein using two-photon excitation assay technology.

We describe the use of fluorophore-doped nanoparticles as reporters in a recently developed ArcDia TPX bioaffinity assay technique. The ArcDia TPX technique is based on the use of polymer microspheres as solid-phase reaction carrier, fluorescent bioaffinity reagents, and detection of two-photon excited fluorescence. This new assay technique enables multiplexed, separation-free bioaffinity assays from microvolumes with high sensitivity. As a model analyte we chose C-reactive protein (CRP). The assay of CRP was optimized for assessment of CRP baseline levels using a nanoparticulate fluorescent reporter, 75 nm in diameter, and the assay performance was compared to that of CRP assay based on a molecular reporter of the same fluorophore core. The results show that using fluorescent nanoparticles as the reporter provides two orders of magnitude better sensitivity (87 fM) than using the molecular label, while no difference between precision profiles of the different assay types was found. The new assay method was applied for assessment of baseline levels of CRP in sera of apparently healthy individuals.

A new technique for multiparameter imaging microscopy: use of long decay time photoluminescent labels enables multiple color immunocytochemistry with low channel-to-channel crosstalk.

In this report, we describe luminescence imaging microscopy using five different photoluminescent dyes in a single specimen. We combined the long decay time luminophores, europium(III) chelate, terbium(III) chelate, palladium(II) coproporphyrin, and platinum(II) coproporphyrin, with a green nuclear stain, Syto 25 trade mark, that emits conventional fast decaying fluorescence. The luminescence emissions from the five different luminophores were separated from each other by the differences in spectra and decay times using time-resolved detection. Applicability of this dye-combination for multiparameter analysis of a biological object was verified in a mixed population of peripheral blood leukocytes. Leukocyte cytocentrifugates were incubated in one step with a cocktail of luminophore-conjugated antibodies recognizing neutrophil- and lymphocyte-specific markers, followed by rapid staining with a mixture of nuclear stain and Pt-porphyrin as an eosinophil stain. The results show that multiple luminescent dyes with long decay time can be used together, and in combination with a conventional fluorophore. The separation of the signals of the long decay time labels was distinctive and enabled reliable identification of different leukocyte types, as well as an automated cell count. The long decay time luminophores together with time-resolved luminescence imaging microscopy (TR-LIM) provide a unique tool for studies of simultaneous expression of multiple antigens at the level of a single cell. In comparison with other multiparameter imaging techniques, the described technique offers increased accuracy of results, simplification of preparation procedure, and dramatic shortening of the total processing time. To our knowledge, this is the first time that simultaneous fivefold labeling/staining and analysis in a single specimen has been performed in the field of immunocytochemistry.

Influence of linker unit on performance of palladium(II) coproporphyrin labelling reagent and its bioconjugates.

In this paper we describe the preparation of a series of new phosphorescent labelling reagents, based on monosubstituted palladium(II) coproporphyrin-I and the isothiocyanato reactive group. The labelling reagents differ with respect to the chemical composition of the linker unit that combines the reactive group and the porphyrin chromophore. Altogether, seven different labelling reagents are prepared. The new labelling reagents are conjugated with monoclonal mouse IgG to yield label conjugates with variable degrees of conjugation. The effect is studied of linker unit on: (a) the conjugation reaction kinetics; (b) the biological activity of the resulting IgG conjugates; and (c) the efficiency of phosphorescence emission. The results show that an increase in the length of the linker unit has a positive effect on both the reactivity of the label and the biological activity of the resulting conjugates. In addition, the results indicate that the labels with the most hydrophilic linker units exhibit the highest phosphorescence emission efficiencies.

Two-photon excitation fluorometric measurement of homogeneous microparticle immunoassay for C-reactive protein.

Recent developments in infrared laser technology have enabled the design of a compact instrumentation for two-photon excitation microparticle fluorometry (TPX). The microparticles can be used in immunoassays as the antibody-coated solid phase to capture an antigen and then detect it with a fluorescently labeled tracer antibody. Unlike most other methods, TPX technology allows low-volume, homogeneous immunoassays with real-time measurements of assay particles in the presence of a moderate excess of fluorescent tracer. In this study, the TPX assay system was used for the reagent characterization and the measurement of C-reactive protein (CRP) in diluted plasma samples, targeting the assay range useful in infectious disease diagnosis. The pentameric structure of the CRP permitted the optimization of an assay with the lowest detectable concentration of 1 microg/L (7.5 pM) by using a single monoclonal antibody both for capture and as the tracer. With a 1:200 predilution of samples, the measurement range of the assay was 1-150 mg/L, but an additional 1:10 dilution was required for higher concentrations. The TPX method showed a good correlation with the reference result obtained in a routine hospital laboratory, demonstrating the feasibility of the technology for immunodiagnostic applications.

Phosphorescent metalloporphyrins as labels in time-resolved luminescence microscopy: effect of mounting on emission intensity.

In this study, we present an investigation of the effects of mounting media on the phosphorescence of metalloporphyrin stained microscopy samples. The samples were: (1) Platinum(II) coproporphyrin (=PtCP) stained porous Sephadex beads; (2) compact polystyrene microspheres coated with IgG-PtCP conjugate; and (3) immunocytochemically labeled human peripheral blood neutrophils. The human neutrophils in a mixed leukocyte population were fixed, permeabilized, and then immunolabeled with PtCP conjugate of monoclonal mouse IgG directed to the intracellular antigen myeloperoxidase. The samples were mounted in twelve different mounting media and studied with quantitative time-resolved luminescence imaging microscopy with respect to the intensity and stability of the phosphorescence signal. The results indicate that microscopy samples stained with PtCP exhibit the brightest phosphorescence emission in non-mounted form or when mounted in non-aqueous permanent mounting media.