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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

Evaluation of the phosphorescent palladium(II)-coproporphyrin labels in separation-free hybridization assays.

Palladium(II)-coproporphyrin label and a set of corresponding monofunctional labeling reagents with different linker arms were evaluated for labeling of oligonucleotides and subsequent use in hybridization assays. The properties of resulting oligonucleotide probes including phosphorescence spectra, quantum yields, lifetimes, and labeling yields were examined as functions of the label and oligonucleotide structures. Upon hybridization with complementary sequences bearing dabcyl, QSY-7, and rhodamine green dyes, the probes displayed strong quenching due to close proximity effects.

Fluorescence-based cell viability screening assays using water-soluble oxygen probes.

A simple luminescence-based assay for screening the viability of mammalian cells is described, based on the monitoring of cell respiration by means of a phosphorescent water-soluble oxygen probe that responds to changes in the concentration of dissolved oxygen by changing its emission intensity and lifetime. The probe was added at low concentrations (0.3 microM to 0.

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.

Performance evaluation of the phosphorescent porphyrin label: solid-phase immunoassay of alpha-fetoprotein.

Phosphorescent conjugates of antibodies, neutravidin, and biotin (pentylamine derivative) were synthesized using previously described monofunctional labeling reagent of platinum(II) coproporphyrin-I with isothiocyanate reactive group (PtCP-NCS). These conjugates, which can be considered as standard reagents for a range of bioanalytical applications, were evaluated in solid-phase immunoassay schemes with the clinical analyte a-fetoprotein (AFP). A custom-designed time-resolved phosphorescence plate reader based on a compact and low-cost 532-nm laser and optimized for measurement of porphyrin labels was used.

Synthesis and evaluation of phosphorescent oligonucleotide probes for hybridisation assays.

Monofunctional, p-isothiocyanatophenyl-derivatives of platinum (II)-coproporphyrin-I (PtCP-NCS) were evaluated as phosphorescent labelling reagents for synthetic oligonucleotides containing a 3'- or 5'-amino modification. Synthesis and purification conditions were optimised to generate high yields and purity of PtCP-labelled oligonucleotide probes. Phosphorescent properties of the PtCP label have been shown to be largely unaffected by conjugation to oligonucleotides of various length, GC composition and label attachment site.

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.