Digoxigenin Explained

Digoxigenin (DIG) is a steroid found exclusively in the flowers and leaves of the plants Digitalis purpurea, Digitalis orientalis and Digitalis lanata (foxgloves), where it is attached to sugars, to form the glycosides (e.g. digoxin, lanatoside C).^[1]

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. K_d of the digoxigenin-antibody interaction has been estimated at ~12 nM ^[2] (compare to K_d~0.1pM for the biotin-streptavidin interaction^[3]).

DIG-binding proteinsTinberg 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.^[4]

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.^{[5] [6]} 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.^[7]
• peptide-DIG conjugates, i.e. bradykinin assay by very sensitive chemiluminescence immunoassays.^[8]
• 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.^[9]
• Digoxigenin may be conjugated to sugars to study glycosylation events,^[10] even in biological systems.

See also

• Immunostaining

Notes and References

1. Book: Polya G . Biochemical Targets of Plant Bioactive Compounds . New York . CRC Press . 2003 . 978-0-415-30829-8 .
2. Tetin SY, Matayoshi ED . Measuring antibody affinity and performing immunoassay at the single molecule level . Analytical Biochemistry. 307. 1. 84–91. August 2002 . 10.1016/S0003-2697(02)00011-8. 12137783 .
3. Duan X . Quantification of the affinities and kinetics of protein interactions using silicon nanowire biosensors . Nature Nanotechnology. 2012 . 7 . 6 . 401–407 . 10.1038/nnano.2012.82. 22635097 . 4180882. 2012NatNa...7..401D .
4. Tinberg CE, Khare SD, Dou J, Doyle L, Nelson JW, Schena A, Jankowski W, Kalodimos CG, Johnsson K, Stoddard BL, Baker D . Computational design of ligand-binding proteins with high affinity and selectivity . Nature . 501 . 7466 . 212–216 . September 2013 . 24005320 . 3898436 . 10.1038/nature12443 . 2013Natur.501..212T .
5. Book: Eisel D, Grünewald-Janho S, Krushen B . DIG Application Manual for Nonradioactive in situ Hybridization . 3rd . Penzberg . Roche Diagnostics . 2002 .
6. Hauptmann G, Gerster T . Two-color whole-mount in situ hybridization to vertebrate and Drosophila embryos . Trends in Genetics . 10 . 8 . 266 . August 1994 . 7940754 . 10.1016/0168-9525(90)90008-T .
7. Hart SM, Basu C . Optimization of a digoxigenin-based immunoassay system for gene detection in Arabidopsis thaliana . Journal of Biomolecular Techniques . 20 . 2 . 96–100 . April 2009 . 19503620 . 2685603 .
8. Décarie A, Drapeau G, Closset J, Couture R, Adam A . Development of digoxigenin-labeled peptide: application to chemiluminoenzyme immunoassay of bradykinin in inflamed tissues . Peptides . 15 . 3 . 511–8 . 1994 . 7937327 . 10.1016/0196-9781(94)90214-3 . 19210640 .
9. Mayilo S, Ehlers B, Wunderlich M, Klar TA, Josel HP, Heindl D, Nichtl A, Kürzinger K, Feldmann J . Competitive homogeneous digoxigenin immunoassay based on fluorescence quenching by gold nanoparticles . Analytica Chimica Acta . 646 . 1–2 . 119–22 . July 2009 . 19523564 . 10.1016/j.aca.2009.05.023 . 2009AcAC..646..119M .
10. Goodarzi MT, Rafiq M, Turner G . An improved multiwell immunoassay using digoxigenin-labelled lectins to study the glycosylation of purified glycoproteins . Biochemical Society Transactions . 23 . 2 . 168S . May 1995 . 7672194 . 10.1042/bst023168s .