Search PubMed⌕ Search

SEARCH · Search PubMed

Results for “Digoxigenin”

Search indexed PubMed citations on genomics, clinical trials, systematic reviews and public health. Explore titles, authors and supplied subject terms, then open the PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 451 records · Page 25Linked to original sources
PubMed1996

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. The avidin-D-labelled antibodies were then incubated with biotinylated horseradish peroxidase or with biotinylated alkaline phosphatase. Such preformed enzyme-labelled complexes were subsequently used in the various assays. A 5-8-fold increase in sensitivity was found when the preformed enzyme-labelled antibody-avidin-D complexes were compared to directly enzyme-labelled antibodies or antibody fragments. Furthermore it was shown that ELISA procedures employing digoxigenin-labelled polyclonal antibodies detected by shuttle conjugates were approximately five times more sensitive than biotinylated antibodies detected by avidin-biotin complexes (ABC method). The greatest sensitivity was obtained using antibody-avidin complexes which consisted of two IgG molecules and 4-6 avidin-D molecules.

Alkaline Phosphatase↗
PubMed1993

Colorimetric reverse transcriptase assay for HIV-1.

A colorimetric assay for reverse transcriptase (RT) of the human immunodeficiency virus type 1 (HIV-1) was developed using a double labelled (biotin and digoxigenin) deoxyuridine triphosphate mixture instead of tritiated thymidine triphosphate. After the RT reaction, the newly polymerized strand from oligodeoxythymidylic acid (oligo-dT) contained both biotin and digoxigenin labels. This nucleotide was bound to streptavidin-magnetic beads by the reaction to biotin. At the detection step, an alkaline phosphatase conjugated antibody to digoxigenin was added, followed by the reaction of a colorimetric substrate for this enzyme. This RT assay was comparable to the isotopic RT assay using purified AMV-RT and two strains of HIV grown in cell lines. In addition it was equivalent to the isotopic RT assay for analysis of the time course of in vitro infection of human peripheral blood lymphocytes by HIV-1. The total assay time after the RT reaction step was less than one hour.

Biotin↗
PubMed1995

Synthetic oligonucleotide cocktails as probes for detection of human parvovirus B19.

A cocktail of 10 oligonucleotides selected at intervals along the length of the genome of human parvovirus B19 was labelled enzymically with digoxigenin and chemically with either digoxigenin (DIG) or dinitrophenyl (DNP). Chemical labelling was easier and more practical for the production of large quantities of probe. Pools labelled with either digoxigenin or DNP could detect 10 fg of B19 DNA in a dot blot reaction using an alkaline phosphatase antibody conjugate and colorimetric detection. Formalin fixed tissue from 11 consecutive cases of fetal hydrops were examined by in situ hybridisation (ISH). Both probe cocktails detected human parvovirus B19 DNA in 3 cases, with positive cells in all tissues examined and with equal sensitivity. The DNP pool is significantly cheaper and simpler to produce and could provide an inexpensive reagent suitable for diagnostic detection of viral nucleic acid in histopathological material.

Base Sequence↗
PubMed1992

Localization of dopamine D2 receptor mRNA with non-radioactive in situ hybridization histochemistry.

A digoxigenin-labeled antisense 42-mer oligonucleotide was used for the localization of the dopamine D2 receptor mRNA in the rat brain. The digoxigenin label was identified with alkaline phosphatase conjugated sheep-anti-digoxigenin. In good analogy with the known terminal fields of the dopaminergic system, various nuclei throughout the brain were labeled. Positive in situ hybridization signals were also found in dopamine cell groups of the substantia nigra and ventral tegmental area and in regions where a dopaminergic innervation is controversial, like the cerebellar cortex and the hippocampus. The non-radioactive in situ hybridization procedure described, shows the localization of the dopamine D2 receptor mRNA with a very high contrast and an optimal histological resolution.

Alkaline Phosphatase↗
PubMed2002

Development of nonradioactive microtiter plate assays for nuclease activity.

We have developed two microtiter plate assays for the detection of DNA cleavage by nucleases, using 3'-biotinylated oligonucleotide substrates. In the covalently linked oligonucleotide nuclease assay (CLONA), the biotinylated substrates are phosphorylated at the 5' end to facilitate their covalent immobilization on CovaLink NH plates. The cleavage of the covalently immobilized substrate by nucleases results in biotin release. The uncleaved substrate molecules are detected with an enzyme-avidin conjugate. The affinity-linked oligonucleotide nuclease assay (ALONA) makes use of substrates with a digoxigenin on the 5' end of the 3'-biotinylated DNA strand. The substrate binds specifically to the wells of streptavidin-coated microtiter plates, in which the nuclease reaction takes place. Uncleaved substrate retains the digoxigenin label, which is detected with an enzyme-labeled anti-digoxigenin antibody. We assessed the efficiency of these two assays by measuring S1 nuclease and DNase I activities, and the inhibitory effect of EDTA and aurintricarboxylic acid on the reaction. Both methods are more convenient than the standard radioactive nuclease assay and are suitable for high-throughput screening of potential nuclease inhibitors, nucleases, and catalytic antibodies. The ALONA assay was found to be more sensitive than the CLONA assay, with a performance similar to that of the standard nuclease assay.

Aurintricarboxylic Acid↗
PubMed1991

Localization of elastase and tumor necrosis factor alpha mRNA by non-radioactive in situ hybridization in cultures of alveolar macrophages.

Digoxigenin is a new tool for labeling probes which can be detected with the help of specific antibodies in the cell by indirect or direct immunostaining. In contrast to the biotin-reaction, the advantage of digoxigenin is that it does not appear in animal or human cells in nature. In comparison to radioactive labeling methods it is favorable in terms of short exposure time and precise localization of signals in the cell. In this paper we describe the localization of elastase and tumor necrosis factor alpha (TNF alpha) mRNA by non-radioactive in situ hybridization of rat alveolar macrophages in cell culture after stimulation with welder steam dusts. Using digoxigenin labeled probes the determination of specific mRNA's expression and their precise localization in the cytoplasm of the cell could be achieved within one day.

Animals↗
PubMed1997

Blot analyses and immunocytochemistry of neural antigens with digoxigenylated primary and secondary antibodies.

While the digoxigenin-anti-digoxigenin (DIG) method is currently the preferred tool for non-radioactive in situ hybridization this study extends its application field to Western blotting of proteins and summarizes advantageous properties of digoxigenylated antibodies in immunocytochemistry. An established protocol for the preparation of digoxigenylated primary antibodies is complemented by dot blot analyses confirming the high sensitivity of hapten-anti-hapten techniques based on primary digoxigenylated antibodies. The comparative Western blot analysis of calcium-binding proteins in nervous tissue is used as an example to show the highly specific detection of relevant antigens with unmodified primary antibodies, digoxigenylated secondary antibodies and anti-digoxigenin-peroxidase conjugates. The application of the DIG technology seems to be especially indicated in tissues containing high amounts of endogenous biotin-bearing proteins which might induce false-positive staining in conventional streptavidin/biotin techniques. Finally, the previously shown suitability of digoxigenylated antibodies for different immunocytochemical procedures is completed here by examples for sensitive single immunoperoxidase staining of neural markers in rat brain and for carbocyanine double immunofluorescence labelling of senile plaques in old rhesus monkeys.

Animals↗
PubMed1998

Simultaneous isotopic and nonisotopic in situ hybridization histochemistry with cRNA probes.

In situ hybridization histochemistry is widely used to study gene expression at the mRNA level in tissues and cells. Double label in situ hybridization allows for coexpression studies. We describe a protocol for the simultaneous hybridization of two cRNA probes tagged with and digoxigenin-UTP, respectively, to frozen brain tissue sections. Hybridization signals of digoxigenin-tagged probes appear as purple cytoplasmic staining following detection of digoxigenin residues by an alkaline-phosphatase-(AP)-linked antibody. Signals resulting from hybridization of radiolabeled probes are detected as silver grains overlying cellular profiles in sections coated with autoradiographic emulsion. Grain counting allows for semiquantitatively estimates of the cellular expression levels of transcripts. Suitable cRNA-probes can be derived from linear templates generated by polymerase chain reaction (PCR) using nested primers which contain RNA-polymerase promotor sites. The cRNA-probes are sensitive and allow an application of this protocol to the detection of a wide range of mRNAs of medium or low abundance.

Corpus Striatum↗
PubMed1980

The question of cumulation of digoxin metabolites in renal failure.

Using high-performance liquid chromatography (HPLC) to separate digoxin from its metabolites digoxigenin, digoxigenin-bis-digitoxoside, and digoxigenin-mono-digitoxoside with subsequent quantitation by 125I radioimmunoassay (RIA), we examined the plasma of patients on long-term oral digoxin therapy. Digoxin was also measured by RIA without prior HPLC separation. Nine patients requiring maintenance dialysis and 9 subjects with lesser degrees of renal impairment were studied. Trace amounts of 1 or more of the digoxin metabolites were found in the plasma of all dialysis patients while subjects with lesser degrees of renal failure had either none or only 1 metabolite in trace amounts. The ratio of HPLC digoxin without HPLC was 0.83 +/- 0.12 (SD) in renal failure patients and 1.06 +/- 0.09 in subjects with renal function (p less than 0.01). RIA overestimates the amount of digoxin in plasma of renal failure patients and in them from 6% to 42% of plasma digoxin, as determined by conventional 125I RIA, may represent compounds other than digoxin.

Chromatography, High Pressure Liquid↗
PubMed2000

Immunocytochemical visualization of transfected DNA in cultured cells.

Nonviral transfection is one of the modern methods for the incorporation of foreign genes into cells. This process involves uptake of foreign genetic material by the cell and further trafficking through the cytoplasm to the nucleus. Elucidation of cytoplasmic pathways of transfection complexes can be useful to improve already existing gene delivery systems or to establish new systems. To monitor transfection complexes in the cell during transfection, we elaborated a method for the visualization of transfection complexes by introducing digoxigenin-labelled nucleotides into foreign DNA followed by detection of digoxigenin label with the use of antibodies directed against digoxigenin. This procedure allowed the visualization of DNA in transfection complexes and to monitor these complexes in cells during transfection.

Cell Line, Transformed↗
PubMed1993

Endothelin receptor B expression in the rat and rabbit lung as determined by in situ hybridization using nonisotopic probes.

Endothelins are a family of potent vasoactive peptides. Full-length cDNA clones to human endothelin receptor B (ETB) mRNA were random prime-labeled with nucleotides conjugated to digoxigenin for in situ hybridization. The labeled cDNA was used to probe frozen sections of rat and rabbit lung. Detection of the digoxigenin-labeled probe was accomplished by an antibody-enzyme conjugate, anti-digoxigenin alkaline phosphatase. The location of the antibody-antigen complex was visualized as an enzyme-linked color reaction. The hybridization, washings, and detection steps were performed under stringent conditions. The following cell types of the rat and rabbit lung had abundant positive reaction product to the ETB probe: bronchiolar and bronchial epithelium, endothelium of smooth-muscle--walled vessels, and bronchial and bronchiolar-associated lymphoid tissue. Abundant positive reaction product was also observed in cell populations in the lung parenchyma. Additional studies are being performed to identify those populations. The results of this study suggest that in addition to vasoactivity, endothelins play other important roles in the lung.

Animals↗
PubMed1990

Characterization of an rRNA gene-specific cDNA probe: applications in bacterial identification.

Discontinuous DNA complementary to Escherichia coli 16S + 23S ribosomal RNA was synthesized by random oligonucleotide priming using reverse transcriptase. cDNA generated from native or denatured rRNA template was labelled by incorporation of either [alpha-32P]dCTP or digoxigenin-labelled dUTP during synthesis, followed by template hydrolysis. The specific activity of the radiolabelled cDNA was 10(7)-10(8) c.p.m. (micrograms rRNA template)-1 with 60-92% incorporation after 5 h. The length of the reverse transcript was between 20 and 1140 nucleotides and was unaffected by exclusion of primer. The cDNA probe could detect 3 pg rRNA by quantitative slot blot. In the non-radiolabelling digoxigenin system 3 micrograms template gave 0.5-2.0 micrograms cDNA after 24 h with a length of between 100 and 1225 bases. This probe could detect 50 pg rRNA. Probes were evaluated in the comparison of Pasteurella haemolytica biotypes by hybridization to Southern blots of restriction-endonuclease-digested total DNA. The digoxigenin-labelled probe was used to identify clinical isolates of Campylobacter jejuni to demonstrate its potential use in laboratories requiring high-sensitivity detection without the use of radioisotopes.

Alkaline Phosphatase↗
PubMed1992

Enumeration of subgingival species on primary isolation plates using colony lifts.

This study evaluated the feasibility of using a colony lift method and DNA probes to enumerate bacterial species cultured on primary isolation plates. Fourteen digoxigenin-labeled whole chromosomal DNA probes representing 12 subgingival species were validated by hybridization with colony lifts prepared from 249 reference strains of 51 species grown on Trypticase soy agar plates supplemented with 5% sheep blood. Colonies of reference strains were lifted onto Nytran filters from plates and treated to lyse cells, remove cellular proteins, denature and fix microbial DNA to the filters. Positive reactions were detected with an anti-digoxigenin antibody conjugated to alkaline phosphatase and revealed by bromo-chloro-indolyl phosphate and nitroblue tetrazolium. Cross-reactions were not observed for 13/14 probes, but 2 strains of Streptococcus mitis reacted with the probe to Streptococcus sanguis II. Subgingival plaque samples were taken by means of a sterile curette from mesiobuccal surfaces of teeth present in each of 26 subjects with differing periodontal disease states. Samples were dispersed, diluted, plated and incubated anaerobically for 7 d at 35 degrees C. Colonies were lifted as described above. Filters were cut into sections and hybridized with the 14 digoxigenin-labeled DNA probes. The probes were used to enumerate the test species and the total number of isolates was determined in 711 plaque samples. The colony lift method and DNA probes provided a sensitive, economical and quantitative method for enumerating cultivable microbial species in subgingival plaque samples. In addition, the amplification provided by growing the organisms on agar plates facilitated determination of numbers of organisms in small plaque samples, such as those from healthy sites.

Actinomyces↗
PubMed1990

Detection of porcine parvovirus using nonradioactive nucleic acid hybridization.

Nonradioactive slot blot hybridization assays were established for the detection of porcine parvovirus (PPV), using either a digoxigenin-labeled DNA probe or a biotinylated RNA probe. All probes were prepared from a 3.3-kb Pst1-EcoR1 DNA fragment of the NADL8 isolate of PPV. The sensitivity and specificity of the probes in a slot blot system were evaluated in comparison with a 32P-radiolabeled RNA probe. Using an anti-digoxigenin alkaline phosphatase detection system, at least 1 ng of viral replicative form (RF) DNA, or the equivalent of 100 plaque forming units (PFU) of infectious virus, could be detected by the digoxigenin-labeled DNA probe. When the biotinylated RNA probe and a strepavidin-alkaline phosphatase detection system were employed, 0.1 ng of RF DNA, or the equivalent of 10 PFU of infectious virus, were detected, comparable to the sensitivity of the 32P-radiolabeled RNA probe. Hybridization was not observed with control DNA samples extracted from swine testicle cells, porcine kidney (PK-15) cells, uninfected mixed swine fetal tissue, or from an unrelated DNA virus (pseudorabies virus) infected PK-15 cells. Different isolates of PPV, namely NADL8, NADL2, KBSH, and Kresse, reacted on an equimolar basis in sensitivity and specificity to the biotinlyated probe. Extraction of DNA directly on the filter membrane (direct filter hybridization) was employed in an attempt to reduce processing time by eliminating DNA extraction steps. Direct filter hybridization was indeed less time consuming; it was also comparable in sensitivity and specificity to those methods employing purified DNA.

Animals↗
PubMed1996

A quantitative luminescence assay for nonradioactive nucleic acid probes.

In histochemical work with digoxigenin- or biotin-labeled nucleic acid probes, reproducibility of in situ hybridization depends on accurate measurement of the amount of non-radioactive label being used. We describe a rapid and sensitive assay for nonradioactive label incorporated into nucleic acids employing a luminogenic substrate for alkaline phosphatase, CSPD (disodium 3-(4-methoxyspirol¿1,2-dioxetane-3,2'-(5'-chloro)tricyclo [3.3.1.1(3,7)]decan¿-4-yl)phenyl phosphate). An alkaline phosphatase-antibody conjugate was bound to digoxigenin-labeled nucleic acids spotted on nylon membranes. Light emission from the reaction of the bound alkaline phosphatase with CSPD was measured with a luminometer. This method allows an accurate determination of digoxigenin incorporated into nucleic acid probes in the range of 0.5-500 fmol of nonradioactive label.

Adamantane↗
PubMed1994

Synthesis of a non-radioactive hepatitis B virus DNA probe from human serum by the polymerase chain reaction.

A method for synthesizing probes for detecting hepatitis B virus DNA in serum was developed. It uses DNA extracted from the serum of an hepatitis B virus carrier as target, and digoxigenin-11-dUTP incorporated in DNA sequences during the polymerase chain reaction as tracer. Using a spot hybridization assay, the sensitivity and specificity of the digoxigenin-labelled DNA probe were compared with two standard hepatitis B virus DNA probes, synthesized with cloned hepatitis B virus DNA and labelled either with digoxigenin or 32P by random priming. Data obtained from the three methods showed an excellent correlation. Thus, hepatitis B virus DNA extracted from human serum and labelled by polymerase chain reaction may be considered a suitable alternative to cloned DNA. It provides reliable probes and eliminates the need for facilities and personnel dedicated to the manipulation of clones. These advantages will allow a wider application of hepatitis B virus DNA testing in clinical practice.

Base Sequence↗
PubMed1995

The photoreactive carbohydrate probe, a novel method for detection of lectins.

One of the main difficulties in the research of lectins is the absence of an adequate technique for their specific and routine detection. Here, we present a photoreactive carbohydrate-probe which could help to overcome this problem. The probe was comopsed by joining four segments: (i) a carbohydrate moiety, (ii) the digoxigenin tag, (iii) the photoreactive cross-linker and (iv) the lysyl-lysine backbone. After incubation with lectins in the dark, the probe can be activated and cross-linked to the lectins by illumination. The result is a lectin with covalently incorporated digoxigenin tag. Such a labelled lectin can be easily identified using anti-digoxigenin antibodies in a Western blot technique. This method is of high specificity and sensitivity and enables direct detection of lectins in complex mixtures, even whole cell homogenates.

Carbohydrates↗
PubMed1995

In situ hybridization shows Dmp1 (AG1) to be a developmentally regulated dentin-specific protein produced by mature odontoblasts.

Acidic phosphorylated proteins are prominent constituents of the extracellular matrix of bone and dentin. It has been postulated that they may have important structural and regulatory roles in the process of tissue mineralization. Studies of a cDNA library, prepared from cells of the rat incisor odontoblast-pulp complex of 3 week old Sprague-Dawley rats, led to the identification of a serine-rich acidic protein, designated AG1, which appeared to be a dentin matrix component. In order to determine which cells of the odontoblast-pulp complex were responsible for the making of AG1, in situ hybridization was carried out using digoxigenin-labeled probes. The full length AG1 cDNA was subcloned into the pBluescript vector, which contains two strong promoters, T3 and T7. The sense and antisense complementary RNA (cRNA) hybridization probes were prepared by in vitro transcription using T3 and T7 polymerases in the presence of 11-dUTP. Incisor sections were obtained from rat embryos at days 16, and 20, and newborns at days 2 and 5. No AG1 mRNA was detected in the embryonic sections, but digoxigenin labeling was evident in odontoblasts secreting mineralizing dentin at postnatal days 2 and 5. Sense probes showed no hybridization. Pulp cells, Meckel's cartilage, and alveolar bone were free of hybridization with the antisense probe. Unexpectedly, a low level of digoxigenin staining was seen in the cytoplasm of secretory ameloblasts, but not in the preameloblasts, stratum intermedium or stellate reticulum of the enamel organ. These data show that AG1 expression is regulated developmentally and is restricted to secretory stage mature odontoblasts.

Alveolar Process↗