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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↗

PubMed2001

A system to enhance the sensitivity of digoxigenin-labelled probe: detection of B19 DNA in serum samples.

A highly sensitive dot-blot hybridisation assay for the routine screening of numerous samples is described, using parvovirus B19 as a model. Digoxigenin-labelled B19 DNA probe was constructed by PCR, hybrids were detected by an anti-digoxigenin monoclonal antibody followed by a second step, using anti-mouse antibodies conjugated to an alkaline phosphatase-dextran complex (EnVision, Dako) was carried out. The sensitivity of the assay was evaluated using both colourimetric and chemiluminescent substrates for the alkaline phosphatase and was compared with a dot-blot hybridisation assay using the digoxigenin-labelled probe and a standard detection system. With the colourimetric substrate, the EnVision system was able to detect 10 fg of B19 DNA, while with the chemiluminescent substrate the sensitivity increased by up to 2 fg (6 x 10(2) genome copies). This detection system was shown to increase the sensitivity of the assay compared to the standard colourimetric visualisation for the digoxigenin-labelled probe, which could detect 0.1 pg. On account of its sensitivity and specificity the dot-blot hybridisation assay together with the chemiluminescent substrate for the EnVision detection system was used to analyse 760 serum samples; the same sera were tested for B19 DNA with the standard colourimetric visualisation for the digoxigenin-labelled probe used routinely in the diagnostic laboratory.

DNA Probes↗

PubMed1994

Digoxigenin-ampicillin conjugate for detection of penicillin-binding proteins by chemiluminescence.

This paper describes a highly sensitive new method for the identification of penicillin-binding proteins (PBPs) that is based on the use of an ampicillin-digoxigenin conjugate (DIG-AMP conjugate) which is detected by immunoblotting and chemiluminescence. The sensitivity of chemiluminescence permitted X-ray film exposure times to be decreased to minutes, as opposed to the days or weeks which are requisite when conventionally radiolabeled beta-lactams are used. Coupling of ampicillin to digoxigenin yielded a product containing digoxigenin (detected by chemiluminescence) which also was inhibitory for Staphylococcus aureus and Escherichia coli. Unconjugated digoxigenin at concentrations of up to 100 micrograms/ml was not inhibitory for either organism. For S. aureus the MICs of DIG-AMP (0.7 microgram of conjugated ampicillin per ml) and of free ampicillin (0.5 microgram/ml) were comparable, indicating that ampicillin retained its bioactivity when coupled to digoxigenin. However, for E. coli the MICs of DIG-AMP (70 micrograms of conjugated ampicillin per ml) and of free ampicillin (8 micrograms/ml) were widely disparate, suggesting that the DIG-AMP conjugate was too large and/or hydrophobic to traverse the E. coli outer membrane via porins. DIG-AMP binding assays with E. coli and S. aureus cell envelopes revealed profiles of PBPs similar to those detected with 125I-ampicillin or [3H]penicillin. DIG-AMP binding to PBPs was completely inhibited in competition experiments with free ampicillin or penicillin, supporting the specificity of the DIG-AMP conjugate for PBPs. DIG-AMP thus represents an advantageous alternative to radioactive beta-lactams for the identification and analysis of PBPs.

Ampicillin↗

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↗

PubMed1995

A comparison of digoxigenin and biotin labelled DNA and RNA probes for in situ hybridization.

A number of in situ hybridization protocols using digoxigenin or biotin labelled probes were assessed for viral nucleic acid detection in formalin fixed, paraffin embedded tissue. Single-step detection protocols for biotin labelled probes produced low sensitivity; however, enzyme based one-step detection protocols for digoxigenin probes produced high sensitivity for both RNA and DNA systems. For both probe types, multistep detection protocols produced equally high sensitivity. Use of an enhanced APAAP procedure for digoxigenin labelled probes achieved maximal sensitivity without use of biotin-strep-tavidin reactions. The sensitivity of nucleic acid detection obtained with a digoxigenin labelled probe is comparable to that obtained using biotin. Digoxigenin labelled probes for nucleic acid detection are recommended for tissues with endogenous biotin.

Animals↗

PubMed1980

Positive inotropic action of digoxigenin and sodium pump inhibition: effects of enhanced sodium influx.

A possibility that intracellular Na+ ions available to Na+,K+-adenosine triphosphatase influence the action of digoxigenin to cause sodium-pump inhibition and a positive inotropic effect was examined with isolated left atria of guinea-pig hearts. The positive inotropic action of digoxigenin developed more rapidly when atria were stimulated at 3 Hz than at 1.5 Hz. The rate of development of the positive inotropic action was dependent on the frequency of membrane depolarizations rather than on contractions. Monensin, a known Na+ ionophore, enhanced the rate of development of the positive inotropic action of digoxigenin. Sodium pump activity, as estimated from ouabain-sensitive 86Rb uptake, was inhibited by digoxigenin in a concentration-dependent manner in quiescent atria. The inhibition was enhanced by electrical stimulation which shifted the concentration-inhibition curves to the left. The sensitivity of the sodium pump for digoxigenin was also affected by membrane depolarizations, suggesting a role for intracellular Na+. These data indicate that similar to the cardiac glycosides, the interaction of the aglycone with Na+,K+-adenosine triphosphatase is essential for the development of the positive inotropic action of this agent.

Action Potentials↗

PubMed1994

Cellular uptake of intracerebroventricularly administered biotin- or digoxigenin-labeled antisense oligodeoxynucleotides in the rat.

1. Antisense oligodeoxynucleotides (ODNs) internally labeled with biotin or digoxigenin were injected into the lateral ventricle of rats and the distribution of the labeled ODNs was examined at several timepoints following the intracerebroventricular (icv) injections. The stability of these injected antisense ODNs, which had no backbone modifications, was also studied by performing recovery experiments. 2. The most intense labeling was observed near the injection site, in periventricular areas, and in perivascular regions. Many of the labeled cells appeared to be neurons, and both the cytoplasm and the nuclei were stained. The labeled cells were detected 15 min after icv injection, demonstrating that the antisense ODNs were taken up rapidly by cells in the parenchyma. The digoxigeninated antisense ODNs were presented in both the cytoplasmic and the nuclear fractions of rat brain extracts, however, the levels appeared to be much lower in the nuclear fractions. 3. Antisense ODNs injected into the lateral ventricle seemed to follow the bulk flow of cerebrospinal fluid (CSF), i.e., from the injection site in the lateral ventricle, through the ventricular system, to the subarachnoid spaces and the perivascular spaces. From the ventricular and perivascular spaces, the antisense ODNs diffused into the extracellular space and were taken up by cells. The full-length digoxigeninated antisense ODNs were detectable within cells after only 15 min, indicating their rapid uptake. In addition, the antisense ODNs appeared to be relatively stable in the brain since the full-length digoxigeninated ODNs were still detectable after 4 hr.

Animals↗

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↗

PubMed1995

Automated detection of digoxigenin-labelled B19 parvovirus amplicons by a capture hybridization assay.

An automated method to identify B19 amplicons, directly labelled with digoxigenin during amplification reaction was developed. The labelled amplicons were hybridized with a biotinylated B19 oligo-probe and captured on commercially available test tubes coated with streptavidin. The hybridized amplicons labelled with digoxigenin were detected using anti-digoxigenin Fab fragments conjugated to peroxidase and the colourimetric reaction automatically evaluated as an immunoenzymatic assay. Fifty serum samples were tested by the assay and the results were in accordance with those obtained by Southern blot analysis of amplified products. Due to the high sensitivity, specificity and reproducibility shown, the assay seems to be a practical and reliable test for the diagnosis of B19 infection and can be easily adapted to identify any digoxigenin-labelled amplified product of viral genomes.

Bacterial Proteins↗

PubMed1996

Labelling of haptenic drug with digoxigenin for competitive immunoassay: its application to lesopitron, a new anxiolytic agent.

A new labelling approach of haptenic drugs with digoxigenin for the development of competitive enzyme immunoassay (EIA) is reported. It consists of the covalent linking of the hapten to the preactivated digoxigenin derivative and revealing the immune complexes with anti-digoxigenin Fab fragments coupled to alkaline phosphatase. This approach has been applied to the development of an EIA for the pharmacokinetic study of lesopitron (E-4424), a new anxiolytic agent. The assay involves a solid-phase immobilization of IgG purified from polyclonal antiserum developed against the butylamino derivative of lesopitron covalently linked to bovine serum albumin. The tracer consists of the covalent linking of the same butylamino derivative to digoxigenin-3-O-methylcarbonyl-epsilon-aminocaproic acid N-hydroxysuccinimide ester. The calibration curve for E-4424 from 12.5 to 6400 pg per well displays an ED50 of 34.5 pg per well, a slope factor of 0.86 and a minimum detectable dose of 4.1 pg per well. The accuracy and the precision of the assay assessed at three different concentrations of E-4424 (500, 1000 and 2000 pg ml-1) give a recovery higher than 95% and intra- and inter-assay RSDs lower than 5 and 10%, respectively. The specificity of the assay was demonstrated by a good correlation of the samples analysed by both HPLC and EIA. A kinetic profile of E-4424 in rats following an oral dose of 50 mg kg-1 has also been established.

Animals↗

PubMed2003

Structural mechanism of specific ligand recognition by a lipocalin tailored for the complexation of digoxigenin.

DigA16 is an artificial digoxigenin-binding protein, which was derived from the bilin-binding protein, a lipocalin of Pieris brassicae, via reshaping of its natural ligand pocket. Here we report the crystal structures of DigA16 in the presence of either digoxigenin or digitoxigenin and for the apo-protein at resolutions below 1.9A. As a consequence of the altogether 17 amino acid substitutions within the binding site significant structural changes have occurred in the four loops that form the entrance to the ligand pocket on top of the structurally conserved beta-barrel framework. For example, one loop adopts a new alpha-helical backbone structure, which seems to be induced by few critical side-chain contacts. Digoxigenin becomes almost fully buried (by 95%) upon complexation, whereby specificity for the hydrophilic steroid is maintained through hydrogen-bonding networks and shape complementarity. The differential binding of the related steroid digitoxigenin is mainly governed by an internal histidine residue, whose side-chain undergoes significant induced fit. Among those amino acids that line the ligand pocket two tyrosine and one tryptophan residue provide the largest contacts. Interestingly, corresponding three side-chains are found with the same mutual orientation in the anti-digoxigenin antibody 26-10, even though the hapten orientation is quite different there and only 66% of the steroid surface is buried in the combining site. Hence, in the case of the engineered lipocalin DigA16 an example of convergent in vitro evolution is observed. Generally, the remarkable structural plasticity of the loop region and the role of polar residues in the binding site illustrate the potential of the lipocalin scaffold for the generation of specific receptor proteins towards a variety of ligands.

Animals↗

PubMed2001

Non-radioactive digoxigenin DNA labeling and immunologic detection of HSV PCR products.

BACKGROUND: Herpes simplex virus (HSV) is a common cause of human skin and mucous membrane infections, and also causes sporadic meningoencephalitis. As a new method for rapid HSV diagnostics, polymerase chain reaction (PCR) has been introduced in clinical laboratories. Radioactive labeling of DNA probes has become a common practice in experimental laboratories. To avoid radioactive labeling of HSV oligonucleotide probes or PCR products, non-radioactive compounds, which are easily detected by enzyme or immunoassay techniques, are introduced. OBJECTIVES: The aim of our study was (1) to introduce non-radioactive labeling of HSV DNA probe by digoxigenin-labeled dUTP; (2) to establish a rapid and reliable laboratory method for rapid HSV diagnostics; (3) to compare the PCR method with the standard virology techniques, such as cell culture virus isolation and HSV direct fluorescent antibody test (DFA). STUDY DESIGN: We have tested the efficiency of PCR method and non-radioactive labeling of HSV DNA probe for detection of HSV from 30 clinical specimens (skin and mucous membrane swabs). HSV was detected in the specimens by standard virology techniques and PCR. Replicated HSV DNA was non-radioactively labeled by random incorporation of digoxigenin-labeled deoxyuridine triphosphate (DIG-dUTP), and the hybrids were detected by the antibody conjugates and the appropriate enzyme-mediated staining reaction (DIG DNA labeling and detection kit non-radioactive, Boehringer Mannheim GmbH). RESULTS: Non-radioactive labeling of hybridization DNA probes with digoxigenin-dUTP was obtained. HSV DNA was successfully multiplied and detected in the HSV-infected cell culture supernatant; however, it was not detected in the clinical specimen supernatant or sediment. HSV DNA was detected by direct PCR method in non-centrifugated clinical specimens. CONCLUSIONS: The PCR method could be successfully used for diagnoses of HSV infections. Since the sensitivity of this method is partly limited by the virus quantity in the specimen, we recommend cultivating the virus in the cell culture at least 24 h prior to PCR. The use of non-radioactive labeling of hybridization DNA probes, such as random primed DNA labeling with digoxigenin-dUTP, has proven both sensitive and specific, and more appropriate for diagnostic purposes than radioactive DNA labeling to be used until standardized commercial tests appear.

Culture Media↗

PubMed2006

Production of anti-digoxigenin antibody HRP conjugate for PCR-ELISA DIG detection system.

There are several methods used to visualize the end product of polymerase chain reactions. One of these methods is an ELISA-based detection system (PCR-ELISA) which is very sensitive and can be used to measure the PCR products quantitatively by a colorimetric method. According to this technique, copies of DNA segments from genomic DNA are amplified by PCR with incorporation of digoxigenin-11-dUTP. Samples are analyzed in a microtiter plate format by alkaline denaturation and are hybridized to biotinylated allele-specific capture probes bound to streptavidin coated plates. Use of the produced anti-digoxigenin antibody horseradish peroxidase conjugate and the substrate 2,2'-azino-di-3-ethylbenzthiazolinsulfonate (ABTS) detected the hybridized DNA. One of the key components in this procedure is the anti-digoxigenin antibody HRP conjugate. Described here is the preparation, purification, and characterization of anti-digoxigenin antibody HRP conjugate for use in the PCR-ELISA DIG detection system. Several biochemical protocols and modifications were applied to increase the sensitivity and specificity of this conjugate for an efficient and cost-effective product.

Animals↗

PubMed1999

Digoxigenin-labeled probe for rapid identification of nisinogenic Lactococcus lactis strains.

From the nisZ gene sequence, a non-radioactive digoxigenin-labeled DNA probe, was tested for detection of nisin-producing strains using polymerase chain reaction amplification. The digoxigenin-labeled DNA probe clearly discriminated between nisin-producing and non-producing strains with a high degree of sensitivity and specificity. By agarose gel electrophoresis, 1.4 ng of nisin DNA was detected using the digoxigenin-labeled DNA probe compared with 11 ng using direct polymerase chain reaction amplification. A colony hybridization method using digoxigenin-labeled DNA to selectively detect nisinogenic bacteria showed that the nis-probe was specific and did not react with any other non-bacteriocinogenic and non-nisinogenic strains.

DNA Probes↗

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↗