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)

Keßler, Christoph; Höltke, Hans-Joachim; Seibl, R.; Burg, Josef; Mühlegger, Klaus

doi:10.1515/bchm3.1990.371.2.917

Cited by 122 publications

(38 citation statements)

References 44 publications

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“…Briefly, 50 J.LI of hybridization solution (2 x SSC, 50% formamide, 1% Denhardt's, 0.1% SDS, 20 mM NaH 2 P0 4 , 20 mM Na 2 HP0 4 , pH 6.8, 100 J.Lg/ml sonicated E. co/i DNA and 15-20 ng/mllabelled probe) was applied to each slide for 16-18 hat 42° C in a moist chamber. Probe L'46 was labelled with dUTP-digoxigenin (Dig) and purified, as described for the kit (Boehringer~Mannheim; see also Kessler et al 1990). Slides were then treated with blocking agent (0.5%) prepared in buffer I (100 mM Tris-HCl, pH 7.5, 150 mM NaCI) for 1 h, incubated for 1-2 h at room temperature with diluted Dig-CIAP (1: 1,000), washed for 2 x 20 min in buffer I and 10 min in buffer III (1 00 mM Tris-HCl, pH 9.5, 100 mM NaCl, 50 mM MgCl 2 ) and finally incubated for 3-4 h in colour developing solution (nitroblue tetrazolium/5-bromo-4-chloro-3-indolyl phosphate) as recommended in the kit.…”

Section: Methodsmentioning

confidence: 99%

Sex chromosome polymorphism and heterogametic males revealed by two cloned DNA probes in the ZW/ZZ fish Leporinus elongatus

et al. 1994

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Abstract. In order to study the divergence of teleost sex chromosomes, subtractive cloning was carried out between genomic DNA ofmales and females ofthe rainbow trout (XX/XY) and of Leporinus elongatus (ZW /ZZ). Inserts cloned in a plasmid vector were individually tested on Southern blots of DNA of males and females for sex specificity. No sex-specific insert was obtained from trout, but two out of ten inserts cloned from L. elongatus showed sex-specific patterns in this species: one corresponds to a sequence present on both Z and W chromosomes, while the other is W specific. Sequences of these two inserts show neither clear homology with other known sequences, nor an open reading frame. They cross-hybridize with the genomic DNA of Leporinusfriderici, but without sex-specific patterns. Twenty-four L. elongatus adults were sexed by gonadal observation, chromosomed examination and Southern hybridization with one or the other insert. Ten males and 11 females had chromosomes and hybridization patterns typical of their sex. One ZW female was recognized as a male with the W-specific probe. This was also the case for two unusual ZW males, one having a male hybridization pattern with the other probe. These three atypical individuals may result from single genetic exchanges between four regions of the Z and the W, giving rise to three atypical W chromosomes. Finding males with such atypical heterochromosomes in a female heterogametic species may indicate that a gradual transition occurs between the heterogametic systems.

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Section: Methodsmentioning

confidence: 99%

Sex chromosome polymorphism and heterogametic males revealed by two cloned DNA probes in the ZW/ZZ fish Leporinus elongatus

et al. 1994

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“…This, as well as the option to complex a wide variety of payloads to different targeting vehicles, makes anti-Dig bispecifics a versatile platform for diagnostic applications and targeted therapy. The Dig-binding module is a humanized derivative of a murine IgG that is applied as an ingredient in laboratory kits and diagnostic assays (15)(16)(17)(18). Availability of its protein structure (Fig.…”

Section: Discussionmentioning

confidence: 99%

“…One well-known hapten is digoxigenin. Coupling reagents/kits for digoxigeninylation are available for many applications (15)(16)(17)(18), including coupling to low molecular weight compounds such as fluorophores, chelating agents, chemotherapeutics, nucleic acids, lipids, nanoparticles, or peptides and proteins (18). Furthermore, antibodies are available that bind digoxigenin with high affinity.…”

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confidence: 99%

Bispecific digoxigenin-binding antibodies for targeted payload delivery

Metz

Haas

Daub

et al. 2011

Proc. Natl. Acad. Sci. U.S.A.

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Bispecific antibodies that bind cell-surface targets as well as digoxigenin (Dig) were generated for targeted payload delivery. Targeting moieties are IgGs that bind the tumor antigens Her2, IGF1R, CD22, or LeY. A Dig-binding single-chain Fv was attached in disulfide-stabilized form to C termini of CH3 domains of targeting antibodies. Bispecific molecules were expressed in mammalian cells and purified in the same manner as unmodified IgGs. They are stable without aggregation propensity and retain binding specificity/affinity to cell-surface antigens and Dig. Digoxigeninylated payloads were generated that retain full functionality and can be complexed to bispecific antibodies in a defined 2∶1 ratio. Payloads include small compounds (Dig-Cy5, Dig-Doxorubicin) and proteins (Dig-GFP). Complexed payloads are targeted by the bispecifics to cancer cells and because these complexes are stable in serum, they can be applied for targeted delivery. Because Dig bispecifics also effectively capture digoxigeninylated compounds under physiological conditions, separate administration of uncharged Dig bispecifics followed by application of Dig payload is sufficient to achieve antibody-mediated targeting in vitro and in vivo.dsFv | imaging | immunotoxin | protein engineering

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“…High-affinity antifluo- (77). Instead of fluorescein, sulfonation of cytosine, introduction of 2-acetylaminofluorene groups into guanine and its derivatives, and incorporation of digoxigen-labeled dUTP into DNA have been used with enzyme-or fluorophore-conjugated antibodies (24,44,77,89,99).…”

Section: Signal Amplification and Detection Methodsmentioning

confidence: 99%

Advances in nucleic acid-based detection methods

Wolcott

1992

Clin Microbiol Rev

151

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Laboratory techniques based on nucleic acid methods have increased in popularity over the last decade with clinical microbiologists and other laboratory scientists who are concerned with the diagnosis of infectious agents. This increase in popularity is a result primarily of advances made in nucleic acid amplification and detection techniques. Polymerase chain reaction, the original nucleic acid amplification technique, changed the way many people viewed and used nucleic acid techniques in clinical settings. After the potential of polymerase chain reaction became apparent, other methods of nucleic acid amplification and detection were developed. These alternative nucleic acid amplification methods may become serious contenders for application to routine laboratory analyses. This review presents some background information on nucleic acid analyses that might be used in clinical and anatomical laboratories and describes some recent advances in the amplification and detection of nucleic acids.

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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)

Cited by 122 publications

References 44 publications

Sex chromosome polymorphism and heterogametic males revealed by two cloned DNA probes in the ZW/ZZ fish Leporinus elongatus

Sex chromosome polymorphism and heterogametic males revealed by two cloned DNA probes in the ZW/ZZ fish Leporinus elongatus

Bispecific digoxigenin-binding antibodies for targeted payload delivery

Advances in nucleic acid-based detection methods

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