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At least 19 recordsLinked to original sources
PubMed1990

Non-radioactive labeling and detection of nucleic acids. I. A novel DNA labeling and detection system based on digoxigenin: anti-digoxigenin ELISA principle (digoxigenin system).

A novel highly sensitive non-radioactive DNA labeling and detection system based on the ELISA principle has been developed. DNA is modified with the cardenolide-hapten digoxigenin by enzymatic incorporation of digoxigenin-labeled deoxyuridine-triphosphate with Klenow enzyme. Digoxigenin is linked to dUTP via an 11-atom linear spacer (Dig-[11]-dUTP). Following hybridization of membrane-bound target-DNA with a digoxigenin-labeled probe, the hybrids are detected by an ELISA reaction using digoxigenin-specific antibodies covalently coupled to the marker enzyme alkaline phosphatase [(Dig):CIAP]. This binding of antibody: marker enzyme-conjugate is followed by an enzyme-catalysed coupled redox reaction with the colour substrates 5-bromo-4-chloro-3-indolyl phosphate (BCIP) and nitroblue tetrazolium salt (NBT) giving rise to a deep-blue coloured, water-insoluble precipitate directly adhering to the membrane. The digoxigenin system allows the detection of 0.1 pg homologous DNA within 16 h in dot- and Southern-blots on nitrocellulose or nylon membranes avoiding any significant background even after a prolonged period of color development. Due to its high sensitivity and specificity, the new system is appropriate for detection of single-copy genes in genomic blots as well as for Northern, slot, colony, plaque and in situ hybridizations.

Animals↗
PubMed1991

The digoxigenin:anti-digoxigenin (DIG) technology--a survey on the concept and realization of a novel bioanalytical indicator system.

A review is given on the novel non-radioactive digoxigenin:anti-digoxigenin (DIG) bioanalytical indicator system. After a general introduction on direct and indirect indicator systems based on previous non-radioactive indicator reactions as well as in vitro and in vivo amplification procedures the principle of the new digoxigenin:anti-digoxigenin technology is demonstrated. The novel system is based on the specific high-affinity interaction between the cardenolide digoxigenin from Digitalis plants and a digoxigenin-specific antibody coupled with a reporter group. A variety of methods for digoxigenin modification of nucleic acids, proteins and glycans are presented. In addition, various applications of the novel non-radioactive indicator system in a variety of direct or indirect detection approaches with either insoluble or soluble substrates are described. It is also shown that with these applications alternative reaction formats are used which are partly characterized by additional amplification steps.

Antibodies↗
PubMed1990

Non-radioactive labeling and detection of nucleic acids. IV. Synthesis and properties of digoxigenin-modified 2'-deoxyuridine-5'-triphosphates and a photoactivatable analog of digoxigenin (photodigoxigenin).

The chemical syntheses of novel digoxigenin-derivatized compounds are described which are modified substrates for enzymatically or photochemically non-radioactive digoxigenin labeling of nucleic acids. Various activated digoxigenin-haptens are coupled to 5-aminoallyl-substituted 2'-deoxyuridine-5'-triphosphate. This results in digoxigenin-modified nucleoside triphosphates of variable spacer lengths (Dig-[4]-dUTP/Dig-[11]-dUTP/Dig-[16]-dUTP) which can be used as substrates for enzymatic labeling of DNA with digoxigenin-haptens by Klenow enzyme-catalysed random-primed synthesis. In addition the synthesis of N-[4-azidobenzoyl]-N'-[(3-O-digoxigeninyl)methylcarbonyl)]-1 ,8-diamino- 3,6-dioxaoctane (photodigoxigenin), a photoactivatable analog of digoxigenin, is described which can be applied for photolabeling of DNA and RNA with digoxigenin-haptens leaving the nucleic acid molecules intact.

DNA↗
PubMed1991

Metabolism of digoxin, digoxigenin digitoxosides and digoxigenin in human hepatocytes and liver microsomes.

In vitro metabolism of digoxin and its cleavage-related compounds was investigated using hepatocytes in primary culture and microsomal fractions both isolated from human livers. On these models, digoxin (DG3) and digoxigenin bisdigitoxoside (DG2) were not shown to be significantly metabolized in vitro in man. Therefore, it appeared that the stepwise cleavage of DG3 and DG2 sugars was not cytochrome P450 dependent. This enzymatic system probably plays a minor role in humans for this particular reaction. However, digoxigenin monodigitoxoside (DG1) and digoxigenin (DG0) which are known to be formed after intra-gastric hydrolysis of DG3, were extensively converted to polar compounds (mainly glucuronides). In addition, using human liver microsomes, a wide variability in UDP-glucuronyl transferase (UDPGT) activities responsible for DG1 glucuronidation was demonstrated. These results suggest that two main factors may contribute to the overall interindividual variability of digoxin biotransformation: 1), the individual intra-gastric pH which influences the sugar cleavage leading to DG1 and DG0; ii), a variability in the level of the hepatic UDPGT specific for digitalis compounds conjugation.

Biotransformation↗
PubMedDate not supplied

Irreversible inhibition of 3H-ouabain binding to Na+ +K+ -ATPase by digoxigenin-3,12-dibromoacetate, an alkylating derivative of digoxigenin.

Digoxigenin-3,12-dibromoacetate (DDB), an alkylating derivation of digoxigenin, was synthesized and tested as a cardiotonic steroid (CS) site directed affinity label for Na+ +K+ -ATPase (ATP phosphohydrolase EC 3.6.1.3). DDB inhibited rat brain Na+ +K+ -ATPase with an I50 of 5 times 10(-6)M and readily displaced specifically bound 3H-ouabain from its binding sites on Na+ +K+ -ATPase. If the enzyme was exposed to DDB prior to the addition of 3H-ouabain its ability to bind 3H-ouabain was decreased, consistent with the concept that DDB interacted irreversibly with the cardiotonic steroid binding sites of Na+ +K+ -ATPase. However, DDB proved to be an even more effective inhibitor of 3H-ouabain binding under conditions where it was unlikely that it could interact with the CS binding sites of this enzyme, suggesting that DDB inhibited 3H-ouabain binding by non-cardiotonic site directed actions. Similarly, the presence of excess strophanthidin did not protect this enzyme against irreversible inhibition by DDB. The data suggest that the presence of a bromoacetate group at the 12 position on cardiotonic steroids does not confer CS binding site directed alkylating properties on these drugs.

Adenosine Triphosphatases↗
PubMed1992

Comparison of 35S- and digoxigenin-labeled RNA and oligonucleotide probes for in situ hybridization. Expression of mRNA of the seminal vesicle secretion protein II and androgen receptor genes in the rat prostate.

The sensitivity of radiolabeled and digoxigenin-labeled RNA probes and synthetic oligonucleotide probes for the detection of seminal vesicle secretion protein II (SVS II) and androgen receptor (AR) mRNA was compared by in situ hybridization in paraformaldehyde-fixed cryostat sections of the rat prostate. Both genes are expressed in different amounts in the various prostatic lobes and contiguous glands. SVS II or AR RNA probes were either labeled with digoxigenin-11-UTP or [35S]UTP by in vitro transcription. A synthetic SVS II oligonucleotide probe was 3' end-labeled (tailed) with either digoxigenin-11-dUTP or [35S]dATP. Hybridized 35S-labeled probes were detected by autoradiography and digoxigenin-labeled probes by immunohistochemistry using alkaline phosphatase conjugated anti-digoxigenin antibody or gold-labeled antibody followed by protein A-gold and silver enhancement. Digoxigenin-labeled probes provided the same degree of sensitivity as their 35S-labeled counterparts for the detection by in situ hybridization of weakly and strongly expressed mRNA. Using both labeling methods, the SVS II RNA probes were more sensitive than the oligonucleotide probes and background labelling of the 35S-labeled oligonucleotide probe was high. The digoxigenin method produced less background with all probe types, hybridization signals showed higher resolution and results were obtained faster than with radiolabeled probes. The immunogold silver enhancement system provided the fastest detection of digoxigenin-labeled probes with a sensitivity and resolution similar to that provided by alkaline phosphatase anti-digoxigenin immunohistochemistry. It is concluded that digoxigenin probe labeling and detection provides a sensitive, reliable, and efficient alternative to radiolabeled probes for in situ hybridization of mRNA.

Animals↗
PubMed1990

Sensitivity of digoxigenin and biotin labelled probes for detection of human papillomavirus by in situ hybridisation.

The sensitivity of digoxigenin and biotin labelled DNA probes for the detection of human papillomavirus (HPV) by dot blotting and in situ hybridisation was compared in tissues from cervical, laryngeal, and anogenital neoplasia. Probes were either labelled with digoxigenin by the random primer technique and detected with anti-digoxigenin antibody, or labelled with biotin by nick translation and detected with streptavidin, both methods having a common final visualisation procedure using alkaline phosphatase. Digoxigenin labelled probes proved two to 10-fold more sensitive by quantitative dot blotting and four-fold more sensitive in detecting HPV 16 DNA in a series of 31 anal carcinomas, compared with biotinylated probes. The digoxigenin method also produced less non-specific background staining of tissue sections than biotin labelled probes. It is concluded that digoxigenin DNA labelling and detection provides a simple, reliable, and efficient alternative to the use of biotin or radioactive isotopes for the detection of HPV DNA by in situ hybridisation. Digoxigenin labelled probes also offer the possibility of double labelling in situ hybridisation procedures when used with biotin labelled probes to provide simultaneous identification of different DNA sequences.

Anus Neoplasms↗
PubMed1989

Detection of human papilloma virus (HPV) DNA in genital biopsy specimens by in situ hybridization with digoxigenin-labeled probes.

The presence of human papillomavirus (HPV) nucleotide sequences in paraffin sections of genital biopsies was examined by in situ hybridization using non-isotopic, digoxigenin-labeled probes representing HPV types 11, 16 and 18. Digoxigenin-labeling of the probes was performed using DNA labeling and a commercially provided detection kit. Hybridization was performed under stringent conditions. The hybrids were detected by using anti-digoxigenin alkaline phosphatase conjugate and visualized with enzyme catalyzed color reaction. In situ hybridization with digoxigenin-labeled probes was a useful technique for identification of HPV infection. The results were compared with the results obtained with radiolabeled DNA probes. The sensitivity of the digoxigenin-labeled probes was equal to the sensitivity of the radiolabeled probes. The background with digoxigenin-labeled probes was very low. Using nonradioactive probes the localization of hybrids at the cellular level was better than 35S-labeled probes.

Autoradiography↗
PubMed1992

[Marking and detection of DNA of leptospires in the dot-blot and situ hybridization with digoxigenin-labelled probes].

DNA of Leptospira interrogans sv. lai strain 017 was labelled with digoxigenin or alpha 32P or biotin and used as probes to detect DNA of leptospires. Probes labelled with digoxigenin were able to detect 0.1-1 pg of homologous DNA and 10(2) of L. interrogans sv. lai strain 017. Probes alpha 32P and biotin detected 1 pg and 10 pg of homologous DNA and 10(3), 5 x 10(3) of L. interrogans sv. lai strain 017 respectively. These three probes couldn't detect L. biflexa sv. patoc strain Patoc I, L. illini strain 3055, Escherichia coli. Bacillus aerogenes capsulalus, Salmonella anatis, K-DNA of Leishmania, and of human WBC. Comparison of digoxigenin-, alpha 32P- and Biotin-labelled probes in the dot-blot hybridization assay on different serogroup sv. of leptospires revealed that digoxigenin-labelled probes were more sensitive than alpha 32P and biotin-labelled probes. The results indicate that digoxigenin-labelled probes DNA can be used for detection of leptospires in field and clinic. We also report procedures in situ hybridization with leptospira in tissues smear and plasma sediment of an experimentally infected guinea pig. It offers the advantage of recognizable of leptospiral morphology in combination with a hybridization signals. The results indicate that digoxigenin-labelled DNA probes of 017 strain might provide tool for routine diagnosis and classification in cases of leptospiral interrogans infection.

Animals↗
PubMed1992

Digoxigenin as an alternative probe labeling for in situ hybridization.

In situ hybridization (ISH) techniques have proven to be invaluable tools for diagnostic molecular laboratories in the detection of gene expression and viral infection in cell and tissue samples. Radioactively labeled probes are still widely used for ISH because of their high sensitivity in detection. Among the non-isotopic systems only biotinylated probes for viral DNA detection found broader acceptance. Recently we introduced the digoxigenin probe labeling and detection system for ISH in our diagnostic molecular laboratory. Our experiences with digoxigenin-labeled probes are summarized in this report in the form of technical recommendations for probe choice, labeling, quantification, hybridization and detection. Several ISH protocols using RNA, DNA and oligonucleotide probes for the detection of mRNA and viral DNA are reported. Advantages, disadvantages and pitfalls of the digoxigenin system as well as comparisons with radiolabeled and other non-isotopic systems are discussed. Digoxigenin-labeled probes provide an attractive alternative to radiolabeled probes and are superior to other nonradioactive probes for ISH. Probe labeling, quantification, hybridization and detection can be performed with low biohazard risk. Working efforts and costs applying digoxigenin-labeled probes or radiolabeled probes for ISH are similar. Digoxigenin-labeled probes are stable for several months, provide an equal sensitivity in detection and a higher degree of cellular resolution than radiolabeled probes. The turnaround time of procedures is short and results of diagnostic ISH can be obtained much quicker than using radiolabeled probes.

Digoxigenin↗
PubMed1979

Differential effect of potassium on the action of digoxin and digoxigenin in guinea-pig heart.

The effect of potassium on the binding of digoxin or digoxigenin to isolated Na+, K+-ATPase was compared with that of potassium on the positive inotropic action of the agents in guinea-pig hearts. The binding of digoxigenin to the enzyme in vitro was reduced to a greater extent by potassium than was the binding of digotoxin. The digoxigenin-induced increase in the force of contraction of left atrial preparations estimated at steady state was reduced at higher potassium concentrations. Potassium had a lesser effect when digoxin was used as the inotropic agent. In contrast, potassium concentrations. Potassium had a lesser effect when digoxin was used as the ininotropic agent. In contrast, potassium reduced the rate of development and also the rate of loss of the positive inotropic action of digoxin observed with left atrial and Langendorff preparations, respectively, to a greater extent than those of digoxigenin. The loss of the positive inotropic effect was more rapid with digoxigenin than with digoxin at each KCl concentration. These data support the contention that the extent of the interaction of digitalis with Na+,K+-ATPase determines the degree of the positive inotropic effect.

Animals↗
PubMed1990

Non-radioactive labeling and detection of nucleic acids. II. Optimization of the digoxigenin system.

The random-primed DNA labeling technique was modified for the incorporation of digoxigenin into DNA as basic component of the digoxigenin-based non-radioactive DNA labeling and detection system. Digoxigenin molecules act as reporter groups for highly sensitive DNA detection by a digoxigenin-specific antibody:alkaline phosphatase-conjugate-catalysed color reaction. The parameters affecting the individual reaction steps of the digoxigenin labeling, hybridization and detection reactions were optimized to maximal sensitivity and specificity.

Blotting, Northern↗
PubMed1989

Detection of cytomegalovirus DNA using probes labelled with digoxigenin.

Cloned HindIII restriction fragments of cytomegalovirus (CMV) DNA (strain AD169) were labelled with biotin, digoxigenin or 32P and used as probes to detect CMV DNA. Probes biotinylated by nick translation, random hexanucleotide priming (RHP) or "photobiotin" were able to detect 10-50 pg of homologous DNA. Probes labelled with digoxigenin or 32P by RHP detected 0.1 pg of homologous DNA, and 1-10 CMV-infected fibroblasts. Comparison of digoxigenin- and 32P-labelled probes in a DNA hybridisation assay on 186 urine specimens demonstrated that these probes were of similar sensitivity, detecting CMV DNA in 40 and 41 specimens, respectively. Positive results were obtained using this hybridisation assay with 11 of 14 specimens (79%) yielding CMV by virus isolation, and with 35 other specimens obtained from patients with laboratory evidence of CMV infection or symptoms compatible with CMV disease. Thus digoxigenin-labelled probes may provide an assay that can detect CMV DNA in specimens yielding a negative result by virus isolation.

Biotin↗
PubMed1990

Detection of Epstein-Barr virus DNA in nasopharyngeal carcinoma using a non-radioactive digoxigenin-labelled probe.

The presence of Epstein Barr virus (EBV) DNA in biopsies from the post-nasal space (PNS) of patients suspected of nasopharyngeal carcinoma (NPC) was detected by in situ cytohybridization with an EBV DNA probe labelled with the novel labelling compound digoxigenin. The digoxigenin probe was hybridised to cryostat sections of NPC biopsies and subsequently detected by an enzyme immunoassay procedure. It was found that in situ cytohybridization using the digoxigenin probe was much more rapid and sensitive (96 h compared to five weeks) than the current method of using 3H-labelled probe. Using the digoxigenin EBV probe, it was found that in all the eighteen NPC biopsies tested, EBV DNA was detected in malignant epithelial cells and infiltrating lymphocytes. EBV DNA was also detected in some normal epithelial cells in these NPC biopsies. EBV DNA was not detected in epithelial cells of non-malignant biopsies.

Cell Line↗
PubMed1991

Digoxigenin-labeled probes for the detection of hepatitis B virus DNA in serum.

A nonradioactive hybridization assay for the detection of hepatitis B virus (HBV) DNA in serum with a digoxigenin-labeled probe is described. The probe was sensitive, being able to detect 0.25 pg of homologous HBV DNA, equivalent to 7 x 10(4) genome copies. After extraction of DNA from clinical samples, the probe detected HBV DNA in 11 of 12 hepatitis B e antigen-positive sera and did not react with 6 hepatitis B surface antigen-negative sera. This result was comparable to that obtained with a radiolabeled probe. When serum samples were treated by the alkaline denaturation method, some false-positive reactions were apparent with the digoxigenin-labeled probe, although their frequency could be reduced to around 8% by modifying the sample treatment with a centrifugation step. Overall, the sensitivity and specificity of the digoxigenin-labeled probe indicate that it is a viable alternative to the radiolabeled probe for the detection of HBV DNA in serum. The lack of radioactive reagents in the digoxigenin labeling and detection system and its long shelf-life make this system suitable for routine use in laboratories.

DNA Probes↗
PubMed1989

Interphase cytogenetics using biotin and digoxigenin labelled probes I: relative sensitivity of both reporter molecules for detection of HPV16 in CaSki cells.

This study was undertaken to develop technology for the detection of nucleic acid using two different DNA probe reporter molecules, the ultimate aim being to differentially label two nucleic acids within the same nucleus. Digoxigenin and biotin were used to label DNA probes. The absolute and relative sensitivity of digoxigenin and biotin labelled DNA probes for detecting integrated human papilloma virus 16 (HPV16) was investigated in CaSki cells by non-isotopic in situ hybridisation (NISH). Several methods for the detection of labelled probes were also investigated. The optimal sensitivity of digoxigenin labelled probe was equivalent to that of biotin when alkaline phosphatase was used as the final detector. The median number of discrete viral signals discernible in each cell with the most sensitive detection system was seven to eight with both labelled probes. The average number of HPV16 genomes in each CaSki cell, derived by dot blot hybridisation, was about 270. The calculated absolute sensitivity of NISH for viral detection in this system is complex because of variation of signal size and number. Nevertheless, one signal per nucleus equates to as little as 30 to 40 viral copies, and probably much less. The ability to distinguish up to 15 discrete signals with both digoxigenin and biotin labelled probes in the nuclei of CaSki cells indicates that these methods will be useful in interphase cytogenetics in material routinely fixed in aldehyde.

Alkaline Phosphatase↗
PubMed1990

Non-radioactive labeling and detection of nucleic acids. III. Applications of the digoxigenin system.

The digoxigenin-based non-radioactive DNA labeling and detection system was applied in various hybridization protocols using digoxigenin-labeled probes obtained by enzymatic incorporation of Dig-[11]-dUTP. In genomic blots single-copy genes (human tissue-type plasminogen activator, constant part of immunoglobulin kappa light chain) can be detected with only 0.5 to 5 micrograms human DNA depending on the type of probe and the length of the hybridizing region. Due to its high sensitivity and specificity, the digoxigenin system is also appropriate for colony-, plaque-, and in situ hybridizations with metaphase chromosome spreads and fixed cells. Especially in the latter applications it is of great advantage, that with the digoxigenin system any significant background or unspecific side reactions with biological materials are avoided.

Blotting, Southern↗