A multiplexed and miniaturized serological tuberculosis assay identifies antigens that discriminate maximally between TB and non-TB sera.

We have developed a multiplexed and miniaturized TB serological assay with the aim of identifying (combinations of) antigens that maximally discriminate between TB and non-TB patients. It features a microarray accommodating 54 TB antigens, less than 1 microl serum consumption and an indirect immunofluorescence detection protocol. With a panel of 20 TB and 80 non-TB sera we ranked combinations of TB antigens with respect to sensitivity and specificity of TB detection by means of logistic step-forward regression analysis.

Enzyme-linked immunosorbent assays using immune complexes for the diagnosis of tuberculosis.

The serodiagnosis of tuberculosis has long been the subject of investigation, but we still lack a test with widespread clinical utility. The poor sensitivity and specificity of commercial assays precludes their use as the sole means of diagnosis. All of these assays use mycobacterial antigens adsorbed onto a surface.

Platinum(II)-based coordination compounds as nucleic acid labeling reagents: synthesis, reactivity, and applications in hybridization assays.

The synthesis, characterization, and molecular interactions of platinum(II) coordination compounds, which contain a distal nonradioactive reporter molecule, with mono- and polynucleotides are described. A [Pt(II)(en)(NH(2)(CH(2))(6)NH-tBoc)Cl](NO(3)) (en=ethylenediamine) entity has been coupled, after removal of the tBoc group, to a number of hapten and fluorophore molecules through succinimide derivatives. The influence of the various tethered reporter groups within these complexes on the reactivity towards guanosine 5'-monophosphate (5'-GMP), as a model for polynucleotide sequences, was investigated to shed light on the use of these reagents in hybridization assays.

Use of horseradish peroxidase- and fluorescein-modified cisplatin derivatives for simultaneous labeling of nucleic acids and proteins.

Background: Microarray platforms will change immunochemical and nucleic acid-based analysis of cell homogenates and body fluids compared with classic analyses. Microarrays use labeled target and immobilized probes, rather than fixed targets and labeled probes. We describe a method for simultaneous labeling of nucleic acids and proteins.

Universal Linkage System: versatile nucleic acid labeling technique.

Over the last two decades nonradioactive nucleic acid labeling and detection systems have overcome the safety, disposal, stability and cost problems that are associated with radioactive techniques. Besides traditional, enzyme-mediated, nonradioactive labeling methods (e.g.

Synthesis and purification of horseradish peroxidase-labeled oligonucleotides for tyramide-based fluorescence in situ hybridization.

A method is presented to conjugate horseradish peroxidase (HRP) to oligodeoxynucleotides for fluorescence in situ hybridization assays employing tyramide signal amplification (TSA). HRP is covalently bound to the oligonucleotide by thiol ether linkage and purified by high-performance liquid chromatography. With TSA detection, a single HRP-labeled oligonucleotide probe is sufficient for in situ detection of clustered DNA repeat sequences with a degree of repetition between 20 and 50.

ULS: a versatile method of labeling nucleic acids for FISH based on a monofunctional reaction of cisplatin derivatives with guanine moieties.

The broad extension of an existing chemical DNA labeling technique for molecular cytogenetics is described. Called the Universal Linkage System (ULS(TM)), it is based on the capability of monoreactive cisplatin derivatives to react at the N7 position of guanine moieties in DNA. Simple repetitive probes, cosmids, PACs, and chromosome-specific painting probes were labeled by ULS and used in a series of multicolor fluorescence in situ hybridization experiments on interphase and metaphase cells.

New strategy for multi-colour fluorescence in situ hybridisation: COBRA: COmbined Binary RAtio labelling.

Multicolour in situ hybridisation (MFISH) is increasingly applied to karyotyping and detection of chromosomal abnormalities. So far 27 colour analyses have been described using fluorescently labelled chromosome painting probes in a so-called combinatorial approach. In this paper a new strategy is presented to use efficiently the currently available number of spectrally separated fluorophores in order to increase the multiplicity of MFISH.

Phosphorescent platinum/palladium coproporphyrins for time-resolved luminescence microscopy.

Streptavidin and antibodies were labeled with phosphorescent platinum and palladium coproporphyrin. The optimal conjugates were selected on the basis of spectroscopic analysis (molar extinction coefficient, quantum yield, lifetime) and using ELISA assays to determine the retention of biological activity and immunospecificity. They were subsequently tested for the detection of prostate-specific antigen, glucagon, human androgen receptor, p53, and glutathione transferase in strongly autofluorescent tissues.

Use of platinum coproporphyrin and delayed luminescence imaging to extend the number of targets FISH karyotyping.

Combinatorial use of fluorophores in multicolor fluorescence in situ hybridization (FISH) allows for the recognition of all human chromosomes. Here we introduce the concept of the use of delayed luminescence labels such as phosphorescent platinum coproporphyrins (PtCP) to extend the number of simultaneously detectable targets in multicolor FISH karyotyping. PtCP-conjugated antibodies were used in combination with conventional FISH labels such as cascade blue, fluorescein, lissamine rhodamine, Cy5, and Cy7.

Fluorescence in situ hybridization using horseradish peroxidase-labeled oligodeoxynucleotides and tyramide signal amplification for sensitive DNA and mRNA detection.

We have used horseradish peroxidase-labeled 40 mer oligodeoxynucleotides (HRP-ODNs) specific for the human cytomegalovirus immediate early gene (HCMV-IE) and a novel dinitrophenol-tyramide signal amplification reagent (DNP-TSA plus) to evaluate their utility in fluorescence in situ hybridization (FISH). For DNA FISH, single or cocktails of HRP-ODNs were hybridized to metaphase chromosomes of rat 9G cells which, as determined by DNA fiber FISH, carry an integrated tandem repeat of 50-60 copies of the HCMV-IE gene. With one layer of DNP-TSA plus deposition and subsequent detection with a fluorochrome-conjugated antibody, four HRP-ODNs were needed to detect the HCMV-IE integration site.

Sensitive mRNA detection by fluorescence in situ hybridization using horseradish peroxidase-labeled oligodeoxynucleotides and tyramide signal amplification.

With the ongoing progress in human genome projects, many genes are discovered whose function and/or expression pattern are not known. Most of these genes are expressed in relatively low abundance compared to housekeeping genes such as elongation factor-1alpha and beta-actin. Gene expression is studied by Northern blot assays or by semiquantitative PCR methods.

Comparative genomic hybridization with lissamine- and fluorescein-labeled nucleotides.

Biotin deoxyuridine 5'-triphosphate (dUTP) and digoxigenin dUTP are the labels most commonly used in comparative genomic hybridization (CGH). The relative infrequent use of direct fluorochrome-labeled nucleotides in CGH is related to the lower sensitivity they provide. Here we report the evaluation of two fluorochrome-conjugated nucleotides that have not been previously used in CGH (lissamine-5-dUTP and fluorescein-N6dATP) and show that this direct label combination performs at least as well as the indirect biotin/digoxigenin pair.

Immunohistochemical visualization of wild-type p53 protein in paraffin-embedded rat liver using tyramide amplification: zonal hepatic distribution of p53 protein after N-hydroxy-2-acetylaminofluorene administration.

P53 protein plays an important role in regulation of the cell cycle. Recently, a role in tumour genesis has also been suggested. The protein is induced after various forms of DNA damage.

Platinum porphyrins as phosphorescent label for time-resolved microscopy.

We investigated phosphorescent metalloporphyrins as potential labels for time-resolved microscopy. On the basis of spectroscopic analysis of their physicochemical properties (quantum yield, molar absorption coefficient, decay times) the best candidates were selected. Next, we synthesized antibody and avidin metalloporphyrin conjugates.

Fluorochrome-labeled tyramides: use in immunocytochemistry and fluorescence in situ hybridization.

The peroxidase-mediated deposition of hapten- and fluorochrome-labeled tyramides has recently been shown to increase the sensitivity of immunofluorescence and fluorescence in situ hybridization techniques. We have evaluated a number of red, green, and blue fluorescent tyramides for detection of antigens in tissue sections and cytospin preparations and for the detection of hapten- and horseradish peroxidase-labeled probes hybridized in situ to cells and chromosomes. With few exceptions, all fluorescent tyramide-based methods provided a considerable increase in sensitivity compared to conventional immunofluorescence and FISH methods.

Influence of fluorochrome labeling density on the photobleaching kinetics of fluorescein in microscopy.

The objective of this study was to identify, through kinetic analysis of individual elementary reactions, the conditions under which a simple first-order photobleaching kinetic model is sufficient for quantitative fluorescence measurements, and those under which more complex photobleaching kinetics must be considered. Three model systems of various fluorophore densities and distributions were employed to verify the kinetic analysis. The results showed that the photobleaching kinetics of free fluorescein at concentrations lower than 5 microM corresponded closely to a single exponential function and therefore involved predominantly simple unimolecular or pseudounimolecular photochemical reactions.

The use of peroxidase-mediated deposition of biotin-tyramide in combination with time-resolved fluorescence imaging of europium chelate label in immunohistochemistry and in situ hybridization.

The application of europium chelates as delayed fluorescent labels in FISH and immunocytochemistry is hampered by their relatively low quantum yield. To increase the intensity of the delayed fluorescence, we have used a recently introduced peroxidase-mediated amplification system. This system results in a large accumulation of biotin-tyramide, which is detected using streptavidin-europium chelate as label.

Improved localization of fluorescent tyramides for fluorescence in situ hybridization using dextran sulfate and polyvinyl alcohol.

Recently, a peroxidase-mediated amplification system has been described for immunofluorescence and fluorescence in situ hybridization studies. It is based on the deposition of hapten- or fluorochrome-labeled tyramide molecules. Although providing a significantly increased detection sensitivity compared to conventional procedures, its localization properties are inferior because of free diffusion of intermediate reaction products before they are immobilized.

Enzyme-labelled antibody-avidin conjugates: new flexible and sensitive immunochemical reagents.

We have prepared avidin-labelled antibodies ('shuttles') with the aim of increasing the sensitivity of detecting mouse IgG and human complement factors in ELISA tests and of detecting monoclonal antibodies and digoxigenin haptens (DIG) in hybridization and immunoblot procedures. Avidin-D was conjugated to goat IgG anti-mouse IgG or to anti-digoxigenin antibodies by thiol/maleimide chemistry. Conjugates of different molecular weight were obtained by Superdex 200 gel filtration.

Horseradish peroxidase-labeled oligonucleotides and fluorescent tyramides for rapid detection of chromosome-specific repeat sequences.

We present a sensitive and rapid fluorescence in situ hybridization (FISH) strategy for detecting chromosome-specific repeat sequences. It uses horseradish peroxidase (HRP)-labeled oligonucleotide sequences in combination with fluorescent tyramide-based detection. After in situ hybridization, the HRP conjugated to the oligonucleotide probe is used to deposit fluorescently labeled tyramide molecules at the site of hybridization.

Ultra-sensitive FISH using peroxidase-mediated deposition of biotin- or fluorochrome tyramides.

We describe a detection principle for indirect fluorescence in situ hybridization (FISH) methods that with only one or two antibody layers dramatically improves FISH signal intensities. The method uses as a first layer an anti-hapten immunoglobulin [or (strept)avidin] conjugated to peroxidase. The quintessence of the method is the use of fluorochrome- or biotin labelled tyramides as peroxidase substrates to generate and deposit many fluorochrome or biotin molecules close to the in situ bound peroxidase.

Quantitation of polymerase chain reaction products by hybridization-based assays with fluorescent, colorimetric, or chemiluminescent detection.

In this report two nonradioactive assays for quantitative analysis of polymerase chain reaction (PCR) products are presented. In the first assay, magnetic beads coated with streptavidin were used to capture biotinylated PCR fragments. After hybridization with a hapten-labeled probe, these beads were analyzed either by flow cytometry (method A) or by immunoenzymatic reactions (method B).

Quantification of sensitive non-isotopic filter hybridizations using the peroxidase catalyzed luminol reaction.

The development of a sensitive, non-isotopic filter hybridization method based on the peroxidase catalyzed luminol reaction is described. High sensitivity was achieved by optimizing the conditions of the hybridization procedure, the immunochemical detection and the peroxidase/luminol reaction. This resulted in the reproducible detection of 10-30 femtogram of target DNA on blots within minutes when a cooled charge coupled device (CCD) camera was used to record the luminescence signal.

3'-end fluorochromized and haptenized oligonucleotides as in situ hybridization probes for multiple, simultaneous RNA detection.

We have used fluorescein-, digoxigenin- and biotin-(di)deoxyXTPs and terminal deoxynucleotidyl transferase for small scale labeling of synthetic oligonucleotide probes and here we show the applicability of such probes for the in situ detection of multiple RNA sequences. The enzymatic 3'-end-labeling methods proved to be good alternatives for the chemical fluorochrome and hapten labeling of 5'-end alkylamino-derivatized oligonucleotides. By combining 3'-end fluorescein-, biotin-, and digoxigenin-labeled oligonucleotides, double and triple hybridizations are feasible.