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PubMed1992

Sensitive chemiluminescent detection of digoxigenin-labeled nucleic acids: a fast and simple protocol and its applications.

A fast and simple protocol for the chemiluminescent detection of digoxigenin-labeled nucleic acids with anti-digoxigenin antibody Fab fragments coupled to alkaline phosphatase and 3-(4-methoxyspiro[1,2-dioxetane-3,2'-tricyclo-[3.3.1.1 (3,7)]decan]-4- yl)phenyl phosphate as substrate is described. The washing and blocking procedure was optimized to yield low background even on positively charged nylon membranes. The sensitivity of the system is equal or better than radioactive methods. Exposure to x-ray or Polaroid film for up to 30 minutes is sufficient for the detection of 70 femtograms of homologous DNA. Human single-copy genes are detected in Southern blots of as low as 0.3 microgram total placental DNA. Blots can be reprobed multiple times very easily. The advantages of the digoxigenin system are high sensitivity, absence of background and ease of reprobing and are illustrated by applications for single-copy gene detection in genomic blots of human DNA, Northern hybridizations to rare mRNA, detection of E. coli genes on blots of genomic digests after pulse field gel electrophoresis, as well as for nonradioactive DNA sequencing blots with digoxigenin-labeled primers.

Actins↗
PubMed1993

[Application of in situ hybridization using digoxigenin--labeled HBV DNA probe and comparison with biotinylated probe].

A method of in situ hybridization for HBV DNA on liver tissue with digoxigenin-labeled probe is described. We have detected the HBV DNA of sixteen liver biopsies. The results showed that the sensitivity and specificity of digoxigenin-labeled probe for in situ hybridization were hig er and stronger then those of biotinylated probe. The practicality of digoxigenin-labeled probe is better. It is an excellent non-radioactive probe for in situ hybridization, which may eventually replace the biotinylated probe. The localization of HBV DNA in hepatocytes could be classified into 4 types, namely, the whole cytoplasmic, focal cytoplasmic, whole nucleic and nuclear membranous-nucleolus. The results of in situ hybridization using digoxigenin-labeled probe could reflect the free or integrated status of HBV genome and the levels of its genomic replication in hepatocytes. The procedure of in situ hybridization using digoxigeninlabeled probe was also discussed.

Biotin↗
PubMed1994

Labeling of basic fibroblast growth factor with digoxigenin: a nonradioactive probe for biochemical and cytological applications.

Digoxigenin, a 391-Da plant sterol, was conjugated to recombinant bFGF with the aim of detecting it with high specificity and sensitivity in cultured eukaryotic cells using antibodies against digoxigenin. The conjugate, bFGF-DIG, displayed a mitogenic activity on endothelial cells equivalent to that of nonlabeled bFGF. Binding of the probe on the cell surface was assessed by ELISA on cells, which allowed discrimination between low- and high-affinity bFGF binding sites. Using a chemiluminescent system, chemical cross-linking of bFGF-DIG with FGF receptors was analyzed directly on Western blots of cell extracts with anti-digoxigenin antibodies. The labeling pattern was identical to that reported with iodinated bFGF, showing that bFGF-DIG bound to the same receptors. The time course of intracellular degradation of internalized bFGF-DIG was also followed by immunodetection on Western blots: the low speed of the catabolic process and the size of the degradation products were comparable to those previously described with iodinated bFGF. In parallel, bFGF-DIG was readily detected by immunofluorescence in cultured cells, and was shown to be an interesting probe to determine bFGF endocytosis pathways by electron microscopy. bFGF-DIG appeared as a multifunctional nonradioactive probe suitable for combined biochemical and cytological studies of bFGF.

Animals↗
PubMed2000

A novel type of receptor protein, based on the lipocalin scaffold, with specificity for digoxigenin.

We demonstrate that the bilin-binding protein, a member of the lipocalin family of proteins, can be structurally reshaped in order to specifically complex digoxigenin, a steroid ligand commonly used for the non-radioactive labelling of biomolecules. 16 amino acid residues, distributed across the four loops which form the binding site of the bilin-binding protein, were subjected to targeted random mutagenesis. From the resulting library the variant DigA16 was obtained by combined use of phage display and a filter-sandwich colony screening assay, followed by in vitro affinity maturation. DigA16 possesses strong binding activity and high specificity for the digoxigenin group, with a K(D) of 30.2(+/-3.6) nM. The derivative compound digitoxigenin is bound even more tightly, with a K(D) of 2.0(+/-0.52) nM, whereas the steroid glycoside ouabain is not recognized at all. Fusion proteins between DigA16 and alkaline phosphatase were constructed and shown to retain both the digoxigenin-binding function and enzymatic activity, irrespective of whether the enzyme was fused to the N or the C terminus of the bilin-binding protein variant. Our findings suggest that the lipocalin scaffold can be generally employed for the construction of specific receptor proteins, so-called "anticalins", which provide a promising alternative to recombinant antibody fragments.

Alkaline Phosphatase↗
PubMed1993

Polymerase chain reaction for synthesis of digoxigenin-labelled DNA probe: application to parvovirus B19 and to polyomavirus BK.

A one-step polymerase chain reaction (PCR) was used to synthesize a digoxigenin-labelled probe, 176 bp long, for the detection of human polyomavirus BK (BKV). A 104 bp-long digoxigenin-labelled probe was generated by 'nested' PCR for the detection of human parvovirus B19 (virus B19). In both cases the whole viral genome was used as template. Different amounts of template as well as different percentages of dTTP substituted by digoxigenin-dUTP (dig-dUTP) in the reaction mixture were employed in order to determine the optimum conditions for the labelled probe synthesis. The sensitivity and the specificity of these PCR-produced probes, together with the simplicity and the reduced time scale of the procedure, suggest the potential of this technique as an additional method for preparing non-radioactive molecular probes for routine diagnosis of viral infections.

BK Virus↗
PubMed1993

Cellular localisation of messenger RNAs in rat testis: application of digoxigenin-labelled ribonucleotide probes to embedded tissue.

In an attempt to identify key changes involved in normal spermatogenesis we have developed methods to enable the study of gene expression by the various subpopulations of testicular cells by use of in-situ hybridisation histochemistry. The use of digoxigenin-labelled ribonucleotide and oligonucleotide probes on testicular tissue perfusion-fixed with Bouin's fixative and embedded in paraffin, polystyrene or methacrylate, has been used to accurately localise three transcripts to three different cell types (Sertoli cells, pachytene spermatocytes, and step 7-12 spermatids) within the seminiferous tubule. The ability to produce semi-thin sections of polystyrene- or methacrylate-embedded tissue and successfully to apply digoxigenin-labelled ribonucleotide or oligonucleotide probes resulted in far greater resolution and unequivocal localisation of mRNA in testicular cells than was previously possible by use of thicker paraffin or frozen sections hybridised with 35S-labelled riboprobes. A comparison of the different embedding media versus digoxigenin-labelled oligonucleotide or ribonucleotide probes is made and we demonstrate the relative sensitivities and merits of each combination.

Animals↗
PubMed1980

Cleavage of digoxigenin digitoxosides by rat liver microsomes.

Microsomal monoxygenases can oxidize the axial hydroxyl of the terminal digitoxosyl of digoxin (dg-3), digoxigenin bis-, and digoxigenin mono-digitoxoside (dg-2 and dg-1, respectively) to an oxo-group. The corresponding metabolites (15'-dehydro-dg-3, 9'-dehydro-dg-2, and 3'-dehydro-1, respectively) have been identified by chromatographic and chemical methods. Only after this oxibation the terminal sugar can be split off, presumably by beta-elimination. Therefore, for the degradation of dg-3 three successive cytochrome P450 catalyzed oxidations are necessary before digoxigenin (dg-0) can be obtained. The highest oxibation rate was observed with dg-1 (120-150 pmoles/mg microsomal protein/min) and by far the lowest with dg-2 (6-7 pmoles/min) as the substrate (concentration was 30/microM). The latter may contribute to the effect that dg-2 is the main dg-3 metabolite in vivo. Pretreatment of rats with canrenoate enhanced the microsomal oxidation of dg-3, dg-2, and dg-1 by a factor of 3.2, 2.3 and 1.3, respectively. In contrast, there was no increase after pretreatment with phenobarbital.

Animals↗
PubMed1990

Nonradioactive labeling of probe with digoxigenin by polymerase chain reaction.

Probes nonradioactively labeled with the steroid hapten digoxigenin have several intriguing properties, including a high sensitivity equivalent to that of radioactive probes, speed in detection, low hazard potential in handling, and possibility of long-term storage. The use of polymerase chain reaction for labeling probe has been demonstrated to offer various advantages including efficient labeling of fragments as small as 100 bp, direct labeling of genomic DNA, and labeling with subnanogram amounts of input DNA. We therefore investigated whether this technique could be adapted for labeling with a relatively large molecule such as digoxigenin. In this report, we show that the polymerase chain reaction is a very efficient technique for synthesis of digoxigenin-labeled DNA and we present an extremely simple procedure for purification of the non-isotopically labeled fragments.

Base Sequence↗
PubMed1994

Pertussis toxin-catalyzed ADP-ribosylation of GTP-binding proteins with digoxigenin-conjugated NAD. Identification of the proteins in plasma membranes and nuclei.

ADP-ribose moiety containing digoxigenin was transferred by pertussis toxin (IAP) to the alpha subunit of Gi (Gi alpha) from digoxigenin-conjugated NAD (DIG-NAD) in a beta gamma subunit-dependent manner. ADP-ribosylation of Gi alpha with DIG-NAD plus IAP was inhibited by native NAD. These results indicate that non-radiolabeled DIG-NAD also serves as the substrate for IAP-catalyzed ADP-ribosylation of G proteins. Using DIG-NAD and fluorescein isothiocyanate-labeled anti-digoxigenin antibody, IAP-sensitive G protein(s) was found to be exist in nuclei as well as plasma membranes of rat liver and HeLa cells. Thus, DIG-NAD is useful to identify pertussis toxin-substrate G proteins.

Adenosine Diphosphate Ribose↗
PubMed1995

In situ hybridization for cytokine mRNA with digoxigenin-labeled riboprobes. Sensitivity of detection and double label applications.

A sensitive in situ hybridization procedure using both digoxigenin and 35S-labeled riboprobes is described that allows detection of single T cells expressing cytokine mRNA species in both single and double label formats. Modifications to existing procedures have been developed that allow in situ hybridization to be performed in either fresh frozen tissue sections or cytocentrifuge preparations of cultured cells. For single label studies, the digoxigenin labeling technique is equivalent to 35S labeling for sensitivity of detection and is superior with respect to precise localization and ease of use. A procedure to detect two cytokine mRNA species in individual cells can be performed using one digoxigenin-labeled riboprobe and one 35S-riboprobe, with equivalent sensitivity between the two labels and no non-specific mixing of the two signals. Since production of many T cell cytokines are controlled by transcriptional mechanisms, the use of in situ hybridization will be useful to investigate the biology of T cell activation, patterns of cytokine phenotype development, and histological localization of cytokine expressing cells in inflammatory lesions. Initial studies using this method to examine cytokine expression by a panel of T cell clones reveals that individual cytokine genes are not necessarily expressed in coordination in individual cells and relatively few individual cells in a Th0 clone express Th1-like and Th2-like cytokines simultaneously.

Animals↗
PubMed1990

Detection of B19 parvovirus infections by a dot-blot hybridization assay using a digoxigenin-labelled probe.

A non-radioactive dot-blot hybridization assay for the detection of B19 parvovirus infections was developed using a digoxigenin-labelled probe both on nylon and nitrocellulose filters. A 700 bp BamHI HindIII fragment of B19 DNA was used to construct the probe. Probe labelling was carried out by incorporating deoxyuridine triphosphate labelled with digoxigenin. The dot-blot hybridization assay was visualized by an immunoenzymatic reaction using antidigoxigenin Fab fragments labelled with alkaline phosphatase. The specificity and sensitivity of digoxigenin-labelled B19 DNA probe was compared with the results obtained with 32P-labelled B19 DNA probe. Out of the 504 serum samples tested, 3 samples were positive in all the hybridization assays performed and 494 were negative, 7 serum samples gave a weak positive reaction when Dig-B19 probe was used on nitrocellulose filters. The 77 pharyngeal swabs tested were negative in all the hybridization assays performed. Our hybridization assay showed a high sensitivity and reproducibility and it appears to be a rapid, practical and reliable test for routine screening of B19 parvovirus DNA in large numbers of clinical specimens.

Adolescent↗
PubMed1993

Chemiluminescent detection of potato and pome fruit viroids by digoxigenin-labeled dot blot and tissue blot hybridization.

A chemiluminescent molecular hybridization protocol was compared to 32P autoradiography for detecting potato spindle tuber viroid (PSTVd) and apple scar skin group viroids (ASSVd). Labeled cRNA probes for PSTVd and ASSVd were synthesized by SP6 RNA polymerase transcription using digoxigenin-11-UTP or alpha-[32P]UTP. Dot blot hybridization of purified viroids and sap extracts from infected plants showed that chemiluminescent detection using digoxigenin-labeled probes was as sensitive as autoradiography using 32P probes. A minimum of 2.0-2.5 pg purified viroid was detected. ASSVd could be detected in as little as 0.4 ng of total nucleic acid extract from infected tissue or in sap extracts diluted to 10(-3) with healthy extracts. Tissue blots of PSTVd-infected potato tubers and tomato roots, stems and leaves and ASSVd-infected apple fruit, stems and petioles, gave positive reactions when hybridized with the digoxigenin probe. No reaction with similar tissues from healthy plants was observed.

Digoxigenin↗
PubMed1991

Detection of HBV-DNA using a digoxigenin-labelled probe. A rapid technique without loss of sensitivity.

The sensitivity of four different non-radiolabelled HBV-DNA probes (digoxigenin-, biotin-, acetylaminoflorene-, chemiprobe-labelled) in detecting HBV-DNA were compared with a 32P-labelled HBV-DNA probe using a spot hybridisation assay. Of the non-radiolabelled probes, the probe using digoxigenin labelling was the most sensitive with reliable and reproducible detection to between 1-3 pg HBV-DNA. The use of digoxigenin-labelled HBV-DNA hybridisation assay deserves wider application for the detection of HBV-DNA because of a number of advantages including ease, speed, safety and cost.

DNA Probes↗
PubMed1992

Detection of hepatitis B virus DNA in human serum samples: use of digoxigenin-labeled oligonucleotides as modified primers for the polymerase chain reaction.

Nonradioactive techniques have been used for the direct detection of hepatitis B virus DNA in human serum samples. A comparison of two different systems using digoxigenin-labeled DNA probes is presented. Furthermore, oligonucleotides containing one molecule of the hapten digoxigenin at the 5'-end were prepared and used as primers for the polymerase chain reaction. Amplified DNA can be directly analyzed with anti-digoxigenin Fab fragments labeled with alkaline phosphatase and chemiluminescent substrates.

Alkaline Phosphatase↗
PubMed1997

Design of non-competitive flow injection enzyme immunoassays for determination of haptens: application to digoxigenin.

A theoretical model immunoradiometric assay (IRMA) was adapted to provide a non-competitive flow injection enzyme immunoassay for haptens and used as a guide in studying the effects of different parameters on the sensitivity, precision and dynamic range of the assay. As well as the concentration of the antibody-enzyme conjugate, the affinity constant, the run time through the affinity column, the homogeneity of the antibody population and purity of the antibody-enzyme conjugate were all shown to be important parameters in the optimisation of the assay. The findings were used to design an optimised enzyme flow injection immunoassay for the model compound digoxigenin in standard solutions. A linear calibration curve was established in the range 0.38-7.7 fmol of digoxigenin, resulting in a precision of 14.8% RSD at 1 fmol and 3.7% RSD at 7.7 fmol. Antibody fragments reacting with digoxigenin and labelled with alkaline phosphatase, (Fab-AP) were used to convert 4-methyl umbelliferyl phosphate to a fluorescent product measured downstream. The sample throughput was 15 h-1 and over 60 injections were possible before regenerating the affinity column.

Chromatography, Affinity↗
PubMed1999

Metabolism of digoxin and digoxigenin digitoxosides in rat liver microsomes: involvement of cytochrome P4503A.

1. The sequential metabolism of digoxin (Dg3) to digoxigenin bis-digitoxoside (Dg2), digoxigenin mono-digitoxoside (Dg1) and digoxigenin (Dg0) was investigated in rat liver microsomes. 2. Kinetic studies produced results consistent with a single enzyme mechanism describing the successive oxidative cleavages. Formation of Dg2 was catalysed with mean (+/-SD) Km and Vmax of 125 +/- 22 microM and 362 +/- 37 pmol/min/mg protein, respectively. The corresponding values for the formation of Dg1 were 61 +/- 5 microM and 7 +/-1 pmol/min/mg protein. Dg0 formation was catalysed with the apparent values of 30 +/- 9 microM and 310 +/- 30 pmol/min/mg protein. 3. Chemical inhibition of cytochrome P450 (CYP) 3A subfamily with ketoconazole and triacetyoleandomycin decreased the formation of Dg2 and Dg1 by up to 90%. Antibodies specific to rat CYP3A2 lowered the rate of oxidative cleavage of Dg3 and Dg2 by up to 85%. Inhibition of CYP2E1, CYP2C subfamily and CYP1A2 by chemical and immuno-inhibition did not affect initial rates of metabolism of Dg3 and Dg2. In contrast, Dg1 metabolism was not affected by triacetyloleandomycin as well as by antibodies to CYP3A2, CYP2C11, CYP2E1, CYP2B1/2B2 and CYP1A2. It was however inhibited by >80% by gestodene and 17alpha-ethynylestradiol (selective inhibitors of human CYP3A). 4. Collectively, these data support the involvement of CYP3A in the cleavage of Dg3 and Dg2 in rat liver microsomes. The enzyme-metabolizing Dg1 remains to be identified.

Animals↗
PubMed1995

In situ hybridization analysis of chromogranin A and B mRNAs in neuroendocrine tumors with digoxigenin-labeled oligonucleotide probe cocktails.

The chromogranin/secretogranin (Cg/Sg) molecules are a family of acidic proteins present in neuroendocrine cells and tumors with secretory granules. They have been frequently used to characterize neuroendocrine cells and tumors by immunohistochemical analyses. Immunoreactivity for CgA is related to the presence of secretory granules in these tumors, so immunohistochemical staining for CgA may be absent in neuroendocrine tumors with only a few secretory granules. RNA in situ hybridization with a series of oligonucleotide probes for CgA and CgB was used to detect the mRNA transcripts for CgA and CgB with digoxigenin-labeled probes in 31 neuroendocrine tumors. These results were compared to ISH with 35S-labeled probes and with immunohistochemical staining for CgA and synaptophysin in the same neoplasms. ISH with 35S-labeled probes for CgA and B detected mRNA transcripts in 31 of 31 tumors, whereas the digoxigenin-labeled probe cocktails for CgA and B were positive in 19 of 31 cases when used separately and in 24 of 31 cases when used together. Immunohistochemical staining for CgA was positive in 22 of 31 cases and for synaptophysin in 23 of 31 cases. The CgA and B oligonucleotide probe cocktails were highly specific, since nonneuroendocrine cells and tumors did not stain and the hybridization signal was abolished by ribonuclease A pretreatment. These results indicate that non-isotopic ISH with digoxigenin-labeled probe cocktails for CgA and B or with 35S-labeled probes can be used in characterizing neuroendocrine cells and tumors in formalin-fixed paraffin-embedded tissue sections even when the CgA protein is not detected by immunohistochemistry.

Base Sequence↗
PubMed1989

Comparative hemodynamic study of a new aminosteroid: LND-623 with its 20 alpha-isomer: LND-369, and with digoxin and digoxigenin-rhamnoside in the anesthetized dog.

Hemodynamic effects of LND-623, a new aminosteroid lacking the C17 lactone ring and the C14 hydroxyl group common to the natural glycosides, were studied in the pentobarbital-anesthetized dog and compared to those of its 20 alpha-isomer LND-369 and of digoxin and digoxigenin-rhamnoside (DRh). Twenty-four mongrel dogs were divided into 4 groups. Group I received either LND-623 or saline on study day 1 and the other drug or saline 1 wk later. Saline was replaced by digoxin in group II, digoxigenin-rhamnoside in group III, and LND-369 in group IV. All drugs except LND-369 were infused as 3.10(-9) mol.kg-1.min-1 over 20 minutes. LND-369 was infused at twice the dose. LND-623 increased left ventricular dP/dt for at least 3 h with a peak at end-infusion or 15 min later, accompanied by a transient vasopressor effect. LND-369 induced, at twice the dose, an inotropic effect of comparable magnitude but of shorter duration. Inversely, it provoked a more marked and prolonged vasopressor effect than its 20 beta-isomer, LND-623. Maximal digoxin inotropic effect occurred later but was of comparable magnitude to that induced by LND-623. Its vasopressor effects reached a plateau rapidly and remained sustained until min 200. Digoxigenin-rhamnoside inotropic but not vasopressor effects are weaker than those of LND-623. It is concluded that LND-623, although lacking the most common structural features of the natural cardiac glycosides, provoked rapid and sustained inotropic activities with transient vasopressor effects. These time-course effects differ from digoxin, and these differences are unrelated to their sugar-moiety characteristics. LND-623 inotropic effect is twice as potent as its 20 alpha-isomer.

Anesthesia↗