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Christoph Kessler - One of the best experts on this subject based on the ideXlab platform.
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The Digoxigenin: Anti-Digoxigenin (DIG) System
Nonradioactive Labeling and Detection of Biomolecules, 1992Co-Authors: Christoph Kessler, Klaus Mühlegger, Rüdiger Rüger, Hans-joachim Höltke, Rudolf Seibl, Josef Burg, Gregor Sagner, G.g. Schmitz, Thomas Walter, Anton HaselbeckAbstract:The Digoxigenin:anti-dioxigenin (DIG) indicator system is based on the specific interaction between the cardenolide steroid DIG, a chemically derived aglycon of digoxin and lanatoside C (see figure), and a high-affinity, DIG-specific antibody (Kessler, 1991).
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the Digoxigenin anti Digoxigenin dig technology a survey on the concept and realization of a novel bioanalytical indicator system
Molecular and Cellular Probes, 1991Co-Authors: Christoph KesslerAbstract:Abstract 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.
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non radioactive labeling of rna transcripts in vitro with the hapten Digoxigenin dig hybridization and elisa based detection
Nucleic Acids Research, 1990Co-Authors: Hans-joachim Höltke, Christoph KesslerAbstract:Abstract We have developed a system for the enzymatic in vitro synthesis of non-radioactively labeled RNA which is derivatized with the hapten Digoxigenin (DIG). The labeling reaction as well as the conditions for hybridization and detection of hybrids by an antibody-conjugate and a coupled colour reaction were analyzed and adapted for high sensitivity and low background. In addition, data on the performance and sensitivity of Digoxigenin-labeled RNA probes in Southern and Northern blots are presented.
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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).
Biological chemistry Hoppe-Seyler, 1990Co-Authors: Klaus Mühlegger, Erasmus Huber, Herbert Von Der Eltz, Rüdiger Rüger, Christoph KesslerAbstract: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.
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NON-RADIOACTIVE LABELING AND DETECTION OF NUCLEIC ACIDS. II, OPTIMIZATION OF THE Digoxigenin SYSTEM
Biological chemistry Hoppe-Seyler, 1990Co-Authors: Hans-joachim Höltke, Klaus Mühlegger, Rudolf Seibl, Josef Burg, Christoph KesslerAbstract: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.
Arne Skerra - One of the best experts on this subject based on the ideXlab platform.
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structural mechanism of specific ligand recognition by a lipocalin tailored for the complexation of Digoxigenin
Journal of Molecular Biology, 2003Co-Authors: Ingo P Korndorfer, Steffen Schlehuber, Arne SkerraAbstract:Abstract 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 1.9 A. 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 β-barrel framework. For example, one loop adopts a new α-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.
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a novel type of receptor protein based on the lipocalin scaffold with specificity for Digoxigenin
Journal of Molecular Biology, 2000Co-Authors: Steffen Schlehuber, Gerald Beste, Arne SkerraAbstract:Abstract 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.
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A novel type of receptor protein, based on the lipocalin scaffold, with specificity for Digoxigenin
Journal of Molecular Biology, 2000Co-Authors: Steffen Schlehuber, Gerald Beste, Arne SkerraAbstract: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. (C) 2000 Academic Press.
G Schmitz - One of the best experts on this subject based on the ideXlab platform.
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sensitive chemiluminescent detection of Digoxigenin labeled nucleic acids a fast and simple protocol and its applications
BioTechniques, 1992Co-Authors: H J Holtke, Gregor Sagner, C Kessler, G SchmitzAbstract: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.
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nonradioactive hla class ii typing using polymerase chain reaction and Digoxigenin 11 2 3 dideoxyuridinetriphosphate labeled oligonucleotide probes
Human Immunology, 1991Co-Authors: Claudia Nevinnystickel, G Schmitz, Maria P Bettinotti, A Andreas, M Hinzpeter, K Muhlegger, E D AlbertAbstract:Abstract We describe a new, simple, rapid, and sensitive nonradioactive technique for the analysis of genetic variations. Genomic DNA was amplified using polymerase chain reaction and amplified DNA was hybridized, with Digoxigenin (DIG)-labeled sequence-specific oligonucleotides. High specificity and sensitivity was achieved when labeling the sequence-specific oligonucleotide at the 3′end with only one DIG using Digoxigenin-11-2′,3′-dideoxy-uridine-5′-triphosphate and DNA deoxynucleotidylexotransferase. The hybridized probes were detected using antiDigoxigenin alkaline phosphatase, fab fragments, and X-phosphate/NBT for visualization. This method was applied to the analysis of HLA-DR4-DRB1 alleles in polymerase chain reaction-amplified genomic DNA and resulted in highly specific and sensitive hybridization signals discriminating even in cases of a one-base-pair mismatch. This technique is particularly suited for HLA oligotyping because it allows the use of tetramethylammonium chloride for the simplification of hybridization and washing conditions.
E D Albert - One of the best experts on this subject based on the ideXlab platform.
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hla class ii typing in a microtitre plate formate using Digoxigenin labelled amplified dna and biotin labelled oligonucleotide probes
European Journal of Immunogenetics, 1993Co-Authors: C Nevinnystickel, E D AlbertAbstract:SUMMARY We describe a new, non-radioactive microtitre plate assay for the analysis of genetic variations at the DNA level. The new method combines hybridization of oligonucleotides with PCR amplified DNA in liquid phase with detection in solid phase using an elisa-reader. Genomic DNA is labelled with Digoxigenin during PCR using a nucleotide mix containing DIG-11-2′-deoxy-uridine-5′-triphosphate (DIG-11-dUTP) DIG labelled, amplified genomic DNA is hybridized in solution with an oligon de which is labelled with one biotin at its 3′-end, using biotin-16,2′,3′-dideoxy ine-5′-triphosphate (BIO-16-ddUTP) and DNA deoxynucleotidylexo-transferase (TdT). The hybridized complex is immobilized in a streptavidin (SA) coated microtitre plate via the biotin and detection of Digoxigenin is performed using anti-Digoxigenin horseradish peroxidase, fab fragments (anti-DIG-POD), and the colorimetric substrate 2,2′-Azino-di-(3-ethylbenzthiazolinsulfonat[6]) (ABTS®). The resulting absorbtion of the assay is analysed in a microtitre plate reader. This method results in highly specific and sensitive hybridization signals and with the 15 oligonucleotides chosen, allows the typing of DR1-DR10.
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nonradioactive hla class ii typing using polymerase chain reaction and Digoxigenin 11 2 3 dideoxyuridinetriphosphate labeled oligonucleotide probes
Human Immunology, 1991Co-Authors: Claudia Nevinnystickel, G Schmitz, Maria P Bettinotti, A Andreas, M Hinzpeter, K Muhlegger, E D AlbertAbstract:Abstract We describe a new, simple, rapid, and sensitive nonradioactive technique for the analysis of genetic variations. Genomic DNA was amplified using polymerase chain reaction and amplified DNA was hybridized, with Digoxigenin (DIG)-labeled sequence-specific oligonucleotides. High specificity and sensitivity was achieved when labeling the sequence-specific oligonucleotide at the 3′end with only one DIG using Digoxigenin-11-2′,3′-dideoxy-uridine-5′-triphosphate and DNA deoxynucleotidylexotransferase. The hybridized probes were detected using antiDigoxigenin alkaline phosphatase, fab fragments, and X-phosphate/NBT for visualization. This method was applied to the analysis of HLA-DR4-DRB1 alleles in polymerase chain reaction-amplified genomic DNA and resulted in highly specific and sensitive hybridization signals discriminating even in cases of a one-base-pair mismatch. This technique is particularly suited for HLA oligotyping because it allows the use of tetramethylammonium chloride for the simplification of hybridization and washing conditions.
Steffen Schlehuber - One of the best experts on this subject based on the ideXlab platform.
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structural mechanism of specific ligand recognition by a lipocalin tailored for the complexation of Digoxigenin
Journal of Molecular Biology, 2003Co-Authors: Ingo P Korndorfer, Steffen Schlehuber, Arne SkerraAbstract:Abstract 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 1.9 A. 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 β-barrel framework. For example, one loop adopts a new α-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.
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a novel type of receptor protein based on the lipocalin scaffold with specificity for Digoxigenin
Journal of Molecular Biology, 2000Co-Authors: Steffen Schlehuber, Gerald Beste, Arne SkerraAbstract:Abstract 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.
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A novel type of receptor protein, based on the lipocalin scaffold, with specificity for Digoxigenin
Journal of Molecular Biology, 2000Co-Authors: Steffen Schlehuber, Gerald Beste, Arne SkerraAbstract: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. (C) 2000 Academic Press.