Digoxigenin

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. K_d of the digoxigenin-antibody interaction has been estimated at ~12 nM ^[3] (compare to K_d~0.1pM for the biotin-streptavidin interaction^[4]).

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 (K_d) of 541 (+/- 193) pM.^[5]

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.^[6][7] 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.^[8]
• peptide-DIG conjugates, i.e. bradykinin assay by very sensitive chemiluminescence immunoassays.^[9]
• 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.^[10]
• Digoxigenin may be conjugated to sugars to study glycosylation events,^[11] even in biological systems.

• Immunostaining