Digoxigenin

Uses in biotechnology

Digoxigenin is a hapten, a small molecule with high antigenicity, that is used in many molecular biology applications similarly to other popular haptens such as 2,4-Dinitrophenol, biotin, and fluorescein. Typically, digoxigenin is introduced chemically (conjugation) into biomolecules (proteins, nucleic acids) to be detected in further assays. Kd of the digoxigenin-antibody interaction has been estimated at ~12 nM (compare to Kd~0.1pM for the biotin-streptavidin interaction).

;DIG-binding proteins Tinberg et al. designed artificial proteins that bind DIG. Their best binder, DIG10.3, was a 141 amino acid protein that bound DIG with a dissociation constant (Kd) of 541 (+/- 193) pM.

Anti-digoxigenin antibodies with high affinities and specificity are used in a variety of biological immuno-assays (e.g. ELISA). The antibodies are labeled with dyes, enzymes or fluorescence, directly or secondarily, for visualization and detection.

Digoxigenin is thus an all-purpose immuno-tag, and in particular a standard immunohistochemical marker for in situ hybridization. In this case it is conjugated to a single species of RNA nucleoside triphosphate (typically uridine), which is then incorporated into RNA (a "riboprobe") as it is synthesized by the cellular machinery.

It allows to make :

• sensitive non-radioactive in situ hybridization probes to detect nucleic acids in plants, able to detect 1 μg of plasmid DNA.
• peptide-DIG conjugates, i.e. bradykinin assay by very sensitive chemiluminescence immunoassays.
• fluorescent and DIG-labeled tracers for competitive immunoassays, i.e. to limit detect digoxin, a drug used to cure cardiac arrhythmia, down to 0.2 ng mL−1.
• Digoxigenin may be conjugated to sugars to study glycosylation events, even in biological systems.

References

References

1. "Digoxigenin". ChemSrc.
2. (2003). "Biochemical Targets of Plant Bioactive Compounds". CRC Press.
3. (August 2002). "Measuring antibody affinity and performing immunoassay at the single molecule level". Analytical Biochemistry.
4. (2012). "Quantification of the affinities and kinetics of protein interactions using silicon nanowire biosensors". Nature Nanotechnology.
5. (September 2013). "Computational design of ligand-binding proteins with high affinity and selectivity". Nature.
6. (2002). "DIG Application Manual for Nonradioactive ''in situ'' Hybridization". Roche Diagnostics.
7. (August 1994). "Two-color whole-mount in situ hybridization to vertebrate and Drosophila embryos". Trends in Genetics.
8. (April 2009). "Optimization of a digoxigenin-based immunoassay system for gene detection in Arabidopsis thaliana". Journal of Biomolecular Techniques.
9. (1994). "Development of digoxigenin-labeled peptide: application to chemiluminoenzyme immunoassay of bradykinin in inflamed tissues". Peptides.
10. (July 2009). "Competitive homogeneous digoxigenin immunoassay based on fluorescence quenching by gold nanoparticles". Analytica Chimica Acta.
11. (May 1995). "An improved multiwell immunoassay using digoxigenin-labelled lectins to study the glycosylation of purified glycoproteins". Biochemical Society Transactions.