Anti-digoxigenin antibodies

Anti-digoxigenin antibody, a Eab fragment (Boehringer Mannheim). 

For identification the lectins must be labeled. The best labels are biotin (streptavidin-enzyme conjugate) or digoxigenin (anti-digoxigenin antibody-enzyme conjugate), but in the case of Concanavalin A, direct labeling with HRP is possible (see below). 

Incubate with streptavidin-HRP conjugate and anti-digoxigenin antibody conjugate for 15-30 min at RT. Wash again thoroughly. Stain as described in Protocols 2.5.4 or 2.5.5. 

In one report, the optical tweezers were used to hold a bend in order to stretch a DNA molecule. First, a DNA molecule (XDNA) was biotin-congugated so that it could be attached to a streptavidin-coated polystyrene bead. The bead was held by optical tweezers inside a PDMS chip. The other end of the DNA was labeled with digoxigenin to be immobilized on the channel coated with anti-digoxigenin antibody. The DNA was stretched by moving the chip, with the bead being held by the optical tweezers [868]. 

Methods have been developed to detect ASOs in plasma and other biological samples, based on hybridization with labeled complementary probes [89], The probes are tagged at one end with biotin and the other end with digoxigenin. After hybridization and binding to neutravidin-coated 96-well plates, nuclease SI is added to degrade unhybridized probe. Anti-digoxigenin antibodies and enzyme-linked secondary antibodies are then added sequentially, followed by enzyme substrate (for example, AttoPhos, which fluoresces after enzymatic cleavage) for detection and quantitation [60, 61]. 

A hybridization-based approach has also been developed to quantitate levels of ribozymes in serum using paired complementary oligonucleotide probes, one labeled with biotin and the other with digoxigenin [90]. The annealed triplex is first collected on streptavidin-coated 96-well plates, then an anti-digoxigenin antibody conjugated with alkaline phosphatase is added, followed by addition of the enzyme substrate p-nitrophenyl phosphate, which is cleaved into a soluble colored product that is quantitated by absorbance at 405 nm. 

Analogously to the O Shanessy method, the glycoproteins are detected with digoxigenin-3-0-succinyl-e-aminocaproic acid hydrazide (Bohringer, now Roche Diagnostics). The glycoproteins are oxidized first with periodate and then transformed with the hydrazide. After SDS gel electrophoresis and blot, they are detected with anti-digoxigenin antibodies coupled to alkaline phosphatase. 

Detection of the hybridization with an anti-digoxigenin-antibody... 

Visualization of the antibody-antigen conjugate by a colour reaction via an alkaline-phosphatase linked to the anti-digoxigenin-antibody... 

A number of molecular biology procedures have also been developed to detect in situ some DNA modifications primarily related to apoptosis and programmed cell death (PCD). These procedures can be collectively referred as in situ end-labeling (ISEL) techniques and are primarily based on the incorporation of digoxigeninated nucleotides into DNA that are subsequently visualized by anti-digoxigenin antibodies that can be conjugated with AP or a fluorochrome [113],... 

Figure 8 Chemiluminescent (A and B) and bioluminescent (C) detections for immobilized hybridizations of PCR product. Dg, digoxigenin Bt, biotin Ad, avidin. Procedure A [30] Biotin moiety is incorporated into PCR products during the amplification reaction, using one 5 -biotinylated primer. The product is hybridized with a Dg-labeled probe and is immobilized on streptavidin-coated magnetic beads. This capture reaction is carried out for 30 min at 37°C. A permanent magnet is used to sediment the beads during washing to remove unbound DNA. By incubation with the washed beads for 45 min at 37°C, anti-Dg antibody conjugated to HRP enzyme is bound to the Dg-labeled probe, and luminol reaction is performed for CL detection. Procedure B [31] Wells of apolystyrene microtiter plate are activated with l-ethyl-3-(3-dimethylaminopropyl)-carbodiimide, and then coated with a labeled cDNA probe complementary to an internal region of the target DNA.

Fig. 30. Detection of mRNA on a membrane or in situ with labeled gene probes. A Detection of mRNA with a fluorescein-labeled single stranded nucleic acid probe, using POD-conjugated anti-fluorescein antibody. B Use of two gene probes labeled with different molecules (fluorescein and digoxigenin) and detected with specific antibodies, both coupled to AP and using two substrates, leading to differently colored products. This in situ hybridization scheme allows the simultaneous detection of two mRNA species in a tissue or cell preparation. C Amplification systems involving more than one antibody can be used to increase specificity and signal intensity.

The labelling of antibodies with the low molecular weight molecules, biotin and digoxigenin (DIG), requires a secondary reagent for their detection and measurement In the case of DIG a labelled anti-DIG antibody is used while fiar biotin, labelled forms of the biotin binding proteins streptavidin and avidin are employed. 

Dig-UTP (BMB) is a UTP analog containing a steroid hapten isolated from Foxglove plants. In the Dig-UTP molecule, digoxigenin is coupled to the C-5 of uracil via an 11-atom spacer arm. Because of the bulky size of the Dig molecule, Dig-UTP labeling of some transcripts is more efficiently performed at a Dig-UTP/ UTP ratio of 0.15/0.85 instead of the usual 0.35/0.65. Dig-labeled probes/products can be detected or quantitated in situ or on membranes by means of an anti-Dig antibody coupled with a reporter enzyme (e.g., alkaline phosphatase) and of the enzymatic reaction with chromogenic or (chemi)luminescent substrates. 

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