Antidigoxigenin antibody

In the method shown in Figure 9B, a firefly luciferase gene is introduced for sensitive bioluminescent detection of target DNA [5], The luciferase-coding DNA requires no posttranslational modification, and the activity of the luciferase produced can be readily measured in the transcription/translation mixture without prior purification. In this assay system, the digoxigenin-labeled probe is first immobilized to polystyrene wells coated with antidigoxigenin antibody. The target... 

Wipe the slide dry around the tissue but keep the tissue area wet. Put onto the each section 200 pL of antidigoxigenin antibody (a Fab fragment), diluted 1 200 in buffer 1. Incubate at least for 2 h at room temperature. 

Do not exceed the incubation time (>2 h) with diluted (1 200) antidigoxigenin antibody solution. 

Drying of the antidigoxigenin antibody. (Always check the surface level of the humidity chamber before the addition of antidigoxigenin antibody solution.)... 

At the time of writing, the antidigoxigenin antibody is available conjugated only to alkaline phosphatase. Peroxidase detection cannot therefore be used with this method. The amplified methods, however, allow greater flexibility. 

To reduce nonspecific binding of antidigoxigenin antibody, cells are preincubated with PBS supplemented with 10% (v/v) fetal calf serum. 

Sheep antidigoxigenin antibody, conjugated to rhodamin, is bought from Boehringer Mannheim (cat. 1207750). It is used at a dilution of 1 10 in PBT. 

Preincubate the cells for 5 min with 100 pd PBS containing 10% (v/v) fetal calf serum. Subsequently, incubate 90 min with 50 pd antidigoxigenin antibody in PBT. Remove unbound antibody by two 5-min washes with 100 pd PBT and two 5-min washes with PBS. 

Several controls should be carried out. (1) To test the specificity of the antidigoxigenin antibody, incubate with replication mixture in which dig-dUTP is replaced by dTTP no labeling should be observed. (2) To make sure that only S-phase cells are labeled, carry out the labeling procedure with cells not in S phase. Note that in a nonsynchronized culture most cells should be unlabeled, because generally only a relatively small fraction of cells is in S phase. 

See also Section III,A,6. If the fluorescence signal is low, an amplification step may be incorporated in the protocol. Use a mouse antidigoxigenin antibody, followed by a dig-conjugated sheep antimouse antibody, followed by a rhodamin-conjugated sheep antidigoxigenin antibody. Wash extensively with PBT between the steps. All these antibodies are available from Boeh-ringer Mannheim. 

Antidigoxigenin antibody/alkaline phosphatase conjugate (available from Boehringer) A1/5000 dilution in Buffer 1 should be made immediately before use (Section 3.2., step 4). 30 mL are required for one 10 x 18 cm filter, 70 mL for 10 filters. 

Enzyme-mediated reporter systems work by catalyzing the precipitation of a visible product at the site of probe hybridization. Antidigoxigenin antibodies are available conjugated to either alkaline phosphatase or horseradish peroxidase (HRP). In this chapter, the enzyme HRP is used to catalyze the oxidation of diarainobenzidine (DAB) to produce a brown precipitate at the site of in situ hybridization (Section 3.3.). 

This chapter describes the use of digoxigenin-labeled probes to detect mRNA transcripts in tissue sections. The sites of hybridization of the probe are visualized using an antidigoxigenin antibody alkaline phosphatase conjugate, and a colorimetric reaction. 

Ferrocene carboxaldehyde 3 and periodate-oxidized digoxigenin were covalently coupled to BSA lysine residues by reductive amination to yield a conjugate behaving as a multivalent antigen. Binding of antidigoxigenin antibody led to a concentration-related decrease of the oxidation current of the ferrocenyl groups [32]. 

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